Tfpi inhibitors and methods of use

ABSTRACT

The invention provides peptides that bind Tissue Factor Pathway Inhibitor (TFPI), including TFPI-inhibitory peptides, and compositions thereof. The peptides may be used to inhibit a TFPI, enhance thrombin formation in a clotting factor-deficient subject, increase blood clot formation in a subject, treat a blood coagulation disorder in a subject, purify TFPI, and identify a TFPI-binding compound.

INCORPORATION BY REFERENCE

This application is a divisional of U.S. patent application Ser. No.13/026,070, filed Feb. 11, 2011, which claims priority to U.S.Provisional Patent Application No. 61/315,758, filed Mar. 19, 2010,which is hereby incorporated by reference in its entirety. The followingapplications also are incorporated by reference in their entirety: U.S.Provisional Patent Application No. 61/139,272, filed Dec. 19, 2008; andU.S. patent application Ser. No. 12/643,818, filed Dec. 21, 2009.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to peptides that bind Tissue FactorPathway Inhibitor (TFPI) and uses thereof.

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: ASCII (text) file named“44241CDIV_SeqListing.txt,” 1,192,996 bytes, created on Mar. 18, 2013.

BACKGROUND OF THE INVENTION

Hemostasis relies on the complex coagulation cascade, wherein a seriesof events mediated by blood clotting factors leads to conversion ofprothrombin to thrombin. Factor X (FX) activation is the central eventof both the intrinsic and extrinsic pathways of the coagulation cascade.The extrinsic pathway has been proposed as the primary activator of thecoagulation cascade (Mackman et al., Arterioscler. Thromb. Case. Biol.,27, 1687-1693 (2007)). Circulating Tissue Factor (TF) and activatedFactor VII (FVIIa) interact to form the “extrinsic complex,” whichmediates activation of FX. The coagulation cascade is amplified by theintrinsic pathway, during which successive activation of factors XII,XI, IX, and VIII results in formation of the “intrinsic” FIXa-FVIIIacomplex that also mediates FX activation. Activated FX promotes thrombinformation, which is required for the body to create fibrin andeffectively curb bleeding.

Severe bleeding disorders, such as hemophilia, result from disruption ofthe blood coagulation cascade. Hemophilia A, the most common type ofhemophilia, stems from a deficiency in factor VIII, while hemophilia Bis associated with deficiencies in Factor IX (FIX). Hemophilia C iscaused by a deficiency in Factor XI (FXI) (Cawthern et al., Blood,91(12), 4581-4592 (1998)). There is currently no cure for hemophilia andother clotting diseases. Factor replacement therapy is the most commontreatment for blood coagulation disorders. However, blood clottingfactors typically are cleared from the bloodstream shortly afteradministration. To be effective, a patient must receive frequentintravenous infusions of plasma-derived or recombinant factorconcentrates, which is uncomfortable, requires clinical settings, isexpensive, and is time consuming. In addition, therapeutic efficacy offactor replacement therapy can diminish drastically upon formation ofinhibitory antibodies. Approximately 30% of patients with severehemophilia A develop inhibitory antibodies that neutralize Factor VIII(FVIII) (Peerlinck and Hermans, Haemophilia, 12, 579-590 (2006)). Fewtherapeutic options exist for patients with anti-Factor antibodies.

Thus, there exists a need in the art for compositions and methods fortreating blood coagulation disorders. The invention provides suchcompositions and methods.

SUMMARY OF THE INVENTION

The invention provides peptides that bind to Tissue Factor PathwayInhibitor (TFPI), including TFPI antagonistic peptides having theability to modulate the blood coagulation cascade. For example, theinvention provides a peptide comprising the amino acid sequenceX₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁ (SEQ ID NO: 3109), wherein

X₇ is selected from the group consisting of L, P, K, S, W, V, N, and Q;

X₈ is selected from the group consisting of L, R, N, F, and I;

X₉ is selected from the group consisting of Y, V, P, and C;

X₁₀ is selected from the group consisting of F, L, and G;

X₁₁ is selected from the group consisting of L, W, V, A, M, T, and S;

X₁₂ is selected from the group consisting of T, F, V, R, A, D, L, E, S,and Y;

X₁₃ is selected from the group consisting of I, M, G, Q, D, and R;

X₁₄ is selected from the group consisting of G, W, Y, L, M, and H;

X₁₅ is selected from the group consisting of N, P, F, H, K, and Y;

X₁₆ is selected from the group consisting of M, D, E, V, G, and K;

X₁₇ is selected from the group consisting of G, I, R, S, T, and L;

X₁₈ is selected from the group consisting of M, K, L, and I;

X₁₉ is selected from the group consisting of Y, G, R, and S;

X₂₀ is selected from the group consisting of A, E, S, C, and Y; and

X₂₁ is selected from the group consisting of A, V, K, and E.

In one aspect, the peptide comprises one or more N-terminal aminoacid(s) directly linked to X₇, wherein the N-terminal amino acid(s)comprise the amino acid sequence selected from the group consisting of

X₆,

X₅X₆,

X₄X₅X₆,

X₃X₄X₅X₆ (SEQ ID NO: 3110),

X₂X₃X₄X₅X₆ (SEQ ID NO: 3111), and

X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 3112), wherein

X₁ is selected from the group consisting of T and G; X₂ is selected fromthe group consisting of F, and V; X₃ is selected from the groupconsisting of V, W, Y, and F; X₄ is selected from the group consistingof D, Q, and S; X₅ is selected from the group consisting of E, T, N, andS; and X₆ is selected from the group consisting of R, H, K, and A.

Alternatively or in addition, the peptide comprises one or moreC-terminal amino acids directly linked to X₂₁, wherein the C-terminalamino acid(s) comprise the amino acid sequence selected from the groupconsisting of

X₂₂,

X₂₂X₂₃,

X₂₂X₂₃X₂₄,

X₂₂X₂₃X₂₄X₂₅ (SEQ ID NO: 3113),

X₂₂X₂₃X₂₄X₂₅X₂₆ (SEQ ID NO: 3114), and

X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇ (SEQ ID NO: 3115), wherein

X₂₂ is selected from the group consisting of Q, I, E, W, R, L, and N;X₂₃ is selected from the group consisting of L, V, M, and R; X₂₄ isselected from the group consisting of K, L, A, and Y; X₂₅ is F; X₂₆ isG; and X₂₇ is T.

In one aspect, the invention provides a peptide comprising the aminoacid sequence set forth in SEQ ID NOs: 1-7, such as a peptide comprisingthe amino acid sequence set forth in any one of JBT0132, JBT0303,JBT0193, JBT0178, JBT0120, and JBT0224, which inhibits TFPI activitywithin the blood coagulation cascade. The invention also provides apeptide that binds TFPI comprising an amino acid sequence of at least60% identity to the sequencePhe-Gln-Ser-Lys-Gly-Asn-Val-Phe-Val-Asp-Gly-Tyr-Phe-Glu-Arg-Leu-Arg-Ala-Lys-Leu(FQSKGNVFVDGYFERLRAKL) (SEQ ID NO: 32).

In addition, the invention provides a peptide that binds TFPI, whereinthe peptide comprises the structure of formula (I):X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019-X1020(SEQ ID NO: 3116). In formula (I),

X1001 is an amino acid selected from the group consisting of Bhf, C, D,F, G, H, I, K, L, M, N, Nmf, Q, R, T, V, W, and Y;

X1002 is an amino acid selected from the group consisting of G, K, andQ;

X1003 is an amino acid selected from the group consisting of A, Aib,Bhs, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

X1004 is an amino acid selected from the group consisting of A, Aib,Bhk, C, D, E, F, G, H, I, K, k, L, M, N, Nmk, P, Q, R, S, T, V, W, andY;

X1005 is an amino acid selected from the group consisting of a, A, Aib,Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T, V, W, andY;

X1006 is an amino acid selected from the group consisting of A, Aib,Btq, C, D, E, F, G, H, I, K, L, M, N, Q, R, S T, V, W, and Y;

X1007 is an amino acid selected from the group consisting of A, F, G, I,K, L, Nmv, P, Q, S, V, W, and Y;

X1008 is an amino acid selected from the group consisting of F, H, K, W,and Y;

X1009 is an amino acid selected from the group consisting of A, Aib, f,I, K, S, T, and V;

X1010 is an amino acid selected from the group consisting of A, Aib, C,D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y;

X1011 is an amino acid selected from the group consisting of Aib, C, K,G, and Nmg;

X1012 is Y;

X1013 is an amino acid selected from the group consisting of A, Aib, C,E, F, G, H, K, L, M, Q, R, W, and Y;

X1014 is an amino acid selected from the group consisting of A, Aib,Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

X1015 is an amino acid selected from the group consisting of(omega-methyl)-R, D, E, K, and R;

X1016 is L;

X1017 is an amino acid selected from the group consisting of(omega-methyl)-R, A, Aib, Bhr, C, Cha, Cit, D, Dab, Dap, E, Eag, Eew, F,G, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, Q, R, S, T, V,W, and Y;

X1018 is an amino acid selected from the group consisting of A, Bal, C,D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y;

X1019 is an amino acid selected from the group consisting of Bhk, K, R,and V; and

X1020 is either present or absent, whereby, in case X1020 is present, itis an amino acid selected from the group consisting of Aib, Bhl, C, F,G, H, I, K, L, Nml, Q, R, S, T, V, W and Y.

In one aspect, the peptide that binds TFPI comprises the structure offormula (III):

X1001-Q-X1003-X1004-X1005-X1006-I/V-X1008-V-X1010-G-Y-C/F-X1014-R-L-X1017-X1018-K-K/L(III) (SEQ ID NO: 3117). In formula (III), X1001, X1003, X1004, X1005,X1006, X1008, X1010, X1014, X1017, and X1018 are each independentlyselected from any amino acid.

The invention further provides a TFPI-binding peptide comprising thestructure of formula (V):X2001-X2002-X2003-X2004-X2005-X2006-[X2007-X2008-X2009-X2010-X2011-X2012-X2013-X2014-X2015-X2016-X2017-X2018]-X2019-X2020-X2021-X2022-X2023(V) (SEQ ID NO: 3118). In formula (V), X2001, X2002, and X2023independently are either present or absent. When present, X2001 is anamino acid selected from the group consisting of A, D, E, F, G, H, I, K,L, P, R, S, T, V, and W; and X2002 is an amino acid selected from thegroup consisting of A, D, E, F, G, H, I, K, L, M, P, R, S, T, V, and W.Additionally,

X2003 is an amino acid selected from the group consisting of A, F, I, K,L, R, S, T, V, W, and Y;

X2004 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, R, S, T, V, and W;

X2005 is W;

X2006 is an amino acid selected from the group consisting of F, H, I, K,L, R V, and

W;

X2007 is an amino acid selected from the group consisting of C, Hcy,Dap, and K, preferably selected from the group consisting of C and Hcy;

X2008 is an amino acid selected from the group consisting of A, G, R, S,and T;

X2009 is an amino acid selected from the group consisting of a, A, I, K,L, M, m, Nle, p, R, and V;

X2010 is an amino acid selected from the group consisting of A, G, I, K,L, P, R, S, T, and V;

X2011 is an amino acid selected from the group consisting of D, E, G, S,and T;

X2012 is an amino acid selected from the group consisting of A, a, D, d,E, e, F, f, G, I, K, k, L, l, M, m, Nle, nle, P, p, R, r, S, s, T, t, V,v, W, and w;

X2013 is an amino acid selected from the group consisting of A, D, d, E,e, F, G, I, K, L, R, S, s, T, V, and W;

X2014 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, M, R, S, T, V, and W;

X2015 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, M, Nle, R, S, T, V, and W;

X2016 is an amino acid selected from the group consisting of A, D, E, F,I, K, L, M, Nle, R, S, T, V, W, and Y;

X2017 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, R, S, T, V, W, and Y;

X2018 is an amino acid selected from the group consisting of C and D(preferably X2018 is C);

X2019 is an amino acid selected from the group consisting of A, F, I, L,S, T, V, and W;

X2020 is an amino acid selected from the group consisting of F and W;

X2021 is an amino acid selected from the group consisting of I, L, andV; and

X2022 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, P, R, S, T, V, and W.

When X2023 is present in the peptide, X2023 is an amino acid selectedfrom the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, W, andY. In one aspect, the peptide comprises a cyclic structure generated bya linkage between X2007 and X2018, indicated in Formula (V) by brackets.

The invention also provides a peptide that binds TFPI, wherein thepeptide comprises at least amino acids 3-22 of the structure of formula(VI):X2001-X2002-F/Y-K-W-F/H-[C-X2008-M/V-X2010-D-X2012-X2013-G-I/T-X2016-S/T-C]-A/V-W-V-X2022-X2023(VI) (SEQ ID NO: 3119). In formula (VI), X2001, X2002 and X2023 are eachindependently present or absent. X2008, X2010, X2012, X2013, X2016, andX2022, as well as X2001, X2002, and X2023 when present, are eachindependently selected from any amino acid. The peptide comprises acyclic structure generated by a linkage between X2007 and X2018,indicated in formula (VI) by brackets.

In one aspect, the invention provides a peptide that binds TFPI, whereinthe peptide comprises at least amino acids 3-21(X3003-X3021) of thestructure of formula (VIII):X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-X3012-X3013-X3014-X3015-X3016-X3017-X3018-X3019-X3020-X3021(VIII) (SEQ ID NO: 3120). In formula (VIII), X3001 and X3002 are eachindependently present or absent. When present, X3001 is an amino acidselected from the group consisting of A, C, D, F, G, I, K, L, M, N, P,Q, R, S, T, W, E, H, and Y; and X3002 is an amino acid selected from thegroup consisting of A, C, D, F, H, K, M, N, P, R, S, T, W, Y, G, I, andL. With respect to the remainder of formula (VIII),

X3003 is an amino acid selected from the group consisting of A, C, D, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y;

X3004 is an amino acid selected from the group consisting of A, C, D, E,F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P;

X3005 is an amino acid selected from the group consisting of C, D, F, G,H, I, K, L, M, N, P, R, S, T, V, W, and Y;

X3006 is an amino acid selected from the group consisting of A, W, C, K,P, R, and H;

X3007 is an amino acid selected from the group consisting of Q, A, C, F,G, H, I, K, L, N, R, S, T, W, and Y;

X3008 is an amino acid selected from the group consisting of A, C, F, G,H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I;

X3009 is an amino acid selected from the group consisting of A, C, F, G,H, I, L, M, R, S, T, V, W, Y, and K;

X3010 is an amino acid selected from the group consisting of A, C, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

X3011 is an amino acid selected from the group consisting of A, G, I, K,L, M, N, Q, R, S, T, V, W, Y, C, F, and H;

X3012 is an amino acid selected from the group consisting of A, C, H, I,K, L, and R;

X3013 is an amino acid selected from the group consisting of A, C, F, G,H, K, L, M, R, S, V, W, Y, and I;

X3014 is an amino acid selected from the group consisting of A, C, F, G,H, I, L, M, N, Q, R, S, T, V, W, Y, and K;

X3015 is an amino acid selected from the group consisting of A, K, andR;

X3016 is an amino acid selected from the group consisting of A, F, K,and R;

X3017 is an amino acid selected from the group consisting of A, C, F, G,I, K, L, N, Q, R, S, T, V, W, Y, H, A, and M;

X3018 is an amino acid selected from the group consisting of A, C, F, I,K, L, M, Q, R, V, W, and Y;

X3019 is an amino acid selected from the group consisting of A, C, D, E,F, G, H, K, L, N, P, Q, R, V, W, Y, and I;

X3020 is an amino acid selected from the group consisting of A, C, F, G,H, K, L, M, N, Q, R, V, W, Y, I, and P; and

X3021 is an amino acid selected from the group consisting of A, C, H, I,K, L, M, N, P, Q, R, T, V, W, Y, F, and G.

Additionally, the invention provides a TFPI-binding peptide comprisingthe structure of formula (IX):X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-H-X3013-X3014-K/R-R-X3017-X3018-X3019-X3020-X3021(IX) (SEQ ID NO: 3121), wherein X3001, X3002, X3003, X3004, X3005,X3006, X3007, X3008, X3009, X3010, X3011, X3013, X3014, X3017, X3018,X3019, X3020, and X3021 are each independently selected from any aminoacid. In addition, the invention includes a peptide that binds TFPI,wherein the peptide comprises an amino acid sequence having at least 60%identity to the sequence of formula (X): Ac-GYASFPWFVQLHVHKRSWEMA-NH2(SEQ ID NO: 223).

The invention further provides a TFPI-binding peptide comprising thestructure of formula (XI):X4001-Q-X4003-X4004-X4005-X4006-X4007-X4008-X4009-X4010-X4011-X4012-X4013-X4014-R-X4016-X4017-X4018-X4019-X4020(XI). With respect to formula (XI),

X4001 is an amino acid selected from the group consisting of F, L, M, Y,1Ni, Thi, Bta, and Dopa;

X4003 is an amino acid selected from the group consisting of C, D, E, M,Q, R, S, T, Ede(O), and Cmc;

X4004 is an amino acid selected from the group consisting of Aib, E, G,I, K, L, M, P, R, W, and Y;

X4005 is an amino acid selected from the group consisting of a, A, Aib,C, D, d, E, G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip, tic,oic, hyp, nma, Ncg, Abg, Apg, thz, and dtc;

X4006 is an amino acid selected from the group consisting of A, C,C(NEM), D, E, G, H, K, M, N, Q, R, S, V, Cit, C(Acm), Nle, I, Ede(O),Cmc, Ed, Eea, Eec, Eef, Nif, and Eew;

X4007 is an amino acid selected from the group consisting of I, V, T,Chg, Phg, and Tle;

X4008 is an amino acid selected from the group consisting of F, H, 1Ni,2Ni, Pmy, and Y;

X4009 is an amino acid selected from the group consisting of Aib, V,Chg, Phg, Abu, Cpg, Tle, and L-2-amino-4,4,4-trifluorobutyric acid;

X4010 is an amino acid selected from the group consisting of A, C, D, d,E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd, and C(NEM);

X4011 is an amino acid selected from the group consisting of A, a, G, p,Sar, c, and hcy;

X4012 is an amino acid selected from the group consisting of Y, Tym,Pty, Dopa, and Pmy;

X4013 is an amino acid selected from the group consisting of C, F, 1Ni,Thi, and Bta;

X4014 is an amino acid selected from the group consisting of A, Aib, C,C(NEM), D, E, K, L, M, N, Q, R, T, V, and Hcy;

X4016 is an amino acid selected from the group consisting of L, Hcy,Hle, and Aml;

X4017 is an amino acid selected from the group consisting of A, a, Aib,C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nva, Opa,Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, and Egt;

X4018 is an amino acid selected from the group consisting of A, Aib,Hcy, hcy, C, c, L, Nle, M, N, and R;

X4019 is an amino acid selected from the group consisting of K, R, andHar; and

X4020 is an amino acid selected from the group consisting of K, L, Hcy,and Aml.

The TFPI-binding peptide of formula (XI) does not comprise the structureformula (XII):X5001-Q-X5003-X5004-X5005-X5006-I/V-X5008-Aib/V-X5010-G-Y-X5013-X5014-R-L-X5017-X5018-K-K/L(XII). In formula (XII),

X5001 is an amino acid selected from the group consisting of F, L, M,and Y;

X5003 is an amino acid selected from the group consisting of C, D, E, M,Q, R, S, and T;

X5004 is an amino acid selected from the group consisting of E, G, I, K,L, M, P, R, W, and Y;

X5005 is an amino acid selected from the group consisting of a, A, Aib,C, D, d, E, G, H, K, k, M, N, Nmg, Q, R, and p;

X5006 is an amino acid selected from the group consisting of A, C, D, E,G, H, K, M, N, Q, R, S, and V;

X5008 is an amino acid selected from the group consisting of F, H, andY;

X5010 is an amino acid selected from the group consisting of A, C, D, E,F, H, D, M, N, P, Q, R, S, T, V, W, and Y;

X5013 is an amino acid selected from the group consisting of Aib, C, andF;

X5014 is an amino acid selected from the group consisting of A, Aib, C,D, E, K, L, M, N, Q, R, T, and V;

X5017 is an amino acid selected from the group consisting of A, Aib, C,Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nve, Opa, Orn, R,and S; and

X5018 is an amino acid selected from the group consisting of A, C, L, M,N, and R.

The invention also includes a peptide consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 4022, 4024,4032, 4036-4047, 4049-4078, 4086-4097, 4100-4127, 4129-4170, 4173-4195,4200-4214, 4217-4225, 4228, 4230, 4231, 4238, and 4239, as well as apeptide consisting of the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1294-1336, 4002, 4013, 4021, 4023, 4025-4031,4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199,4215, 4216, 4226, 4277, 4229, 4232, and 4233.

In the context of the disclosure, any peptide encompassed by any offormulas (I) to (XI) and any TFPI-binding peptide described herein isalso referred to as “the peptide of the invention” and as “a peptide asdescribed herein.”

In some embodiments, the peptide of the invention binds TFPI-1 (e.g.,TFPI-1a) and, optionally, improves TFPI-regulated thrombin generation inthe absence of FVIII, FIX, and/or FXI. A composition (e.g., apharmaceutical composition) comprising the peptide also is provided.

In addition, the invention provides methods of using the peptide of theinvention. For example, the invention provides a method of inhibiting aTFPI comprising contacting the TFPI with a peptide as described herein.The invention also provides a method of enhancing thrombin formation ina clotting factor-deficient subject, a method for increasing blood clotformation in a subject, and a method of treating a blood coagulationdisorder in a subject. The methods are, in their entirety, also referredto herein as, e.g., “the method of the invention.” The methods compriseadministering to the subject a peptide as provided herein in an amounteffective to enhance thrombin formation, an amount effective to enhanceblood clot formation, or an amount effective to treat the bloodcoagulation disorder in the subject. Unless explicitly indicated to thecontrary, the description provided herein with respect to one peptide ofthe invention or method of the invention applies to each and everypeptide of the invention and method of the invention, respectively.Further aspects of the invention include use of the peptide of theinvention for the manufacture of a medicament, a method for targeting acell displaying TFPI, a method for treating or diagnosing a subjectsuffering from a disease or at risk of suffering from a disease, amethod of purifying TFPI, and a method of identifying a TFPI-bindingcompound.

The invention also includes a method for identifying a TFPI-bindingcompound, the method comprising (a) contacting a peptide comprising TFPIKunitz domain 1 (KD1) with a test compound, and (b) detecting binding ofthe test compound to a TFPI binding site defined by KD1 amino acidresidues corresponding to human TFPI residues Phe28, Lys29, Ala30,Asp32, Ile46, Phe47, and Ile55. A method for inhibiting human TFPI, themethod comprising contacting human TFPI with an inhibitor that bindshuman TFPI at a binding site defined by amino acid residues Phe28,Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55, also is provided. Theinvention further provides a computer storage media having computerexecutable instructions that, when executed on the processor of acomputer, implement a method of modeling interaction between selectedthree dimensional (3D) points in a TFPI Kunitz domain 1 (KD1) proteinand a test compound, as well as a method of comparing a test compound toselected three dimensional points in a TFPI Kunitz domain 1 (KD1)protein.

The preceding methods are, in their entirety, also referred to hereinas, e.g., “the method of the invention.”

The following numbered paragraphs each succinctly define one or moreexemplary variations of the invention:

1. A peptide that binds TFPI, comprising the structure of formula (XI):X4001-Q-X4003-X4004-X4005-X4006-X4007-X4008-X4009-X4010-X4011-X4012-X4013-X4014-R-X4016-X4017-X4018-X4019-X4020(XI), wherein X4001 is an amino acid selected from the group consistingof F, L, M, Y, 1Ni, Thi, Bta, and Dopa; wherein X4003 is an amino acidselected from the group consisting of C, D, E, M, Q, R, S, T, Ede(O),and Cmc; wherein X4004 is an amino acid selected from the groupconsisting of Aib, E, G, I, K, L, M, P, R, W, and Y; wherein X4005 is anamino acid selected from the group consisting of a, A, Aib, C, D, d, E,G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip, tic, oic, hyp, nma,Ncg, Abg, Apg, thz, and dtc; wherein X4006 is an amino acid selectedfrom the group consisting of A, C, C(NEM), D, E, G, H, K, M, N, Q, R, S,V, Cit, C(Acm), Nle, I, Ede(O), Cmc, Ed, Eea, Eec, Eef, Nif, and Eew;wherein X4007 is an amino acid selected from the group consisting of I,V, T, Chg, Phg, and Tle; wherein X4008 is an amino acid selected fromthe group consisting of F, H, 1Ni, 2Ni, Pmy, and Y; wherein X4009 is anamino acid selected from the group consisting of Aib, V, Chg, Phg, Abu,Cpg, Tle, and L-2-amino-4,4,4-trifluorobutyric acid; wherein X4010 is anamino acid selected from the group consisting of A, C, D, d, E, F, H, K,M, N, P, Q, R, S, T, V, W, Y, Nmd, and C(NEM); wherein X4011 is an aminoacid selected from the group consisting of A, a, G, p, Sar, c, and hcy;wherein X4012 is an amino acid selected from the group consisting of Y,Tym, Pty, Dopa, and Pmy; wherein X4013 is an amino acid selected fromthe group consisting of C, F, 1Ni, Thi, and Bta; wherein X4014 is anamino acid selected from the group consisting of A, Aib, C, C(NEM), D,E, K, L, M, N, Q, R, T, V, and Hcy; wherein X4016 is an amino acidselected from the group consisting of L, Hcy, Hle, and Aml; whereinX4017 is an amino acid selected from the group consisting of A, a, Aib,C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nva, Opa,Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, and Egt; wherein X4018 is anamino acid selected from the group consisting of A, Aib, Hcy, hcy, C, c,L, Nle, M, N, and R; wherein X4019 is an amino acid selected from thegroup consisting of K, R, and Har; and wherein X4020 is an amino acidselected from the group consisting of K, L, Hcy, and Aml; and whereinthe peptide does not comprise the following structure of formula (XII):X5001-Q-X5003-X5004-X5005-X5006-I/V-X5008-Aib/V-X5010-G-Y-X5013-X5014-R-L-X5017-X5018-K-K/L(XII), wherein X5001 is an amino acid selected from the group consistingof F, L, M, and Y; wherein X5003 is an amino acid selected from thegroup consisting of C, D, E, M, Q, R, S, and T; wherein X5004 is anamino acid selected from the group consisting of E, G, I, K, L, M, P, R,W, and Y; wherein X5005 is an amino acid selected from the groupconsisting of a, A, Aib, C, D, d, E, G, H, K, k, M, N, Nmg, Q, R, and p;wherein X5006 is an amino acid selected from the group consisting of A,C, D, E, G, H, K, M, N, Q, R, S, and V; wherein X5008 is an amino acidselected from the group consisting of F, H, and Y; wherein X5010 is anamino acid selected from the group consisting of A, C, D, E, F, H, D, M,N, P, Q, R, S, T, V, W, and Y; wherein X5013 is an amino acid selectedfrom the group consisting of Aib, C, and F; wherein X5014 is an aminoacid selected from the group consisting of A, Aib, C, D, E, K, L, M, N,Q, R, T, and V; wherein X5017 is an amino acid selected from the groupconsisting of A, Aib, C, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L,M, Nle, Nve, Opa, Orn, R, and S; and wherein X5018 is an amino acidselected from the group consisting of A, C, L, M, N, and R.

2. The peptide according to paragraph 1, wherein X4001 is an amino acidselected from the group consisting of F, Y, 1Ni, Bta, and Dopa; whereinX4003 is an amino acid selected from the group consisting of D, E, andS; wherein X4004 is K; wherein X4005 is an amino acid selected from thegroup consisting of p, Nmg, NpropylG, aze, pip, tic, oic, and hyp;wherein X4006 is an amino acid selected from the group consisting of C,E, K, R, S, V, C(Acm), Nle, C(NEM), I, and Cit; wherein X4007 is V orTle; wherein X4008 is an amino acid selected from the group consistingof H, 1Ni, 2Ni, and Pmy; wherein X4009 is an amino acid selected fromthe group consisting of V, Abu, and Tle; wherein X4010 is an amino acidselected from the group consisting of D, P, C, and T; wherein X4011 isan amino acid selected from the group consisting of G, a, c, hcy, andSar; wherein X4012 is Y; wherein X4013 is an amino acid selected fromthe group consisting of F, 1Ni, and Bta; wherein X4014 is an amino acidselected from the group consisting of Aib, C, E, and Hcy; wherein X4016is an amino acid selected from the group consisting of L, Aml, Hle, andHcy; wherein X4017 is an amino acid selected from the group consistingof A, Aib, C, c, Aic, Eca, and Deg; wherein X4018 is an amino acidselected from the group consisting of A, Aib, C, c, L, and Hcy; whereinX4019 is K; and wherein X4020 is an amino acid selected from the groupconsisting of L, Aml, and Hcy.

3. The peptide according to paragraph 1 or paragraph 2 furthercomprising N-terminal amino acid(s) and/or moieties linked to X4001 andselected from the group consisting of FAM-Ttds, PE, Palm, 2-phenylacetyl, 3-phenyl propionyl, 2-(naphtha-2-yl)acetyl, hexanoyl, 2-methylpropionyl, 3-methyl butanoyl, 2-naphthylsulfonyl, and1-naphthylsulfonyl.

4. The peptide according to any one of paragraphs 1-3 further comprisingX4021 linked to X4020, wherein X4021 comprises C-terminal amino acid(s)and/or moieties selected from the group consisting of C, c, C(NEM),K(Ttds-maleimidopropionyl(EtSH)), FA19205, FA19204, FA19203, FA03202,K(Tdts-maleimid), K(AOA), and Cea.

5. The peptide according to any one of paragraphs 1-4, wherein thepeptide comprises a cyclic structure.

6. The peptide according to paragraph 5, wherein the cyclic structure isformed between X4018 and X4021.

7. The peptide according to paragraph 6, wherein (a) X4018 is C or c and(b) X4021 is Cea.

8. The peptide according to paragraph 5, wherein the cyclic structure isformed between X4011 and X4014.

9. The peptide according to paragraph 8, wherein (a) X4011 is c or hcyand (b) X4014 is C or Hcy.

10. The peptide according to any one of paragraphs 1-9, comprising anintramolecular disulfide bond.

11. The peptide according to any one of paragraphs 1-10, wherein theIC₅₀ of the peptide is less than 1000 nM.

12. The peptide according to any one of paragraphs 1-10, wherein theIC₅₀ of the peptide is less than 250 nM.

13. The peptide according to any one of paragraphs 1-10, wherein theIC₅₀ of the peptide is less than 50 nM.

14. The peptide according to any one of paragraphs 1-10, wherein theIC₅₀ of the peptide is less than 10 nM.

15. A peptide consisting of the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 4022, 4024, 4032, 4036-4047, 4049-4078,4086-4097, 4100-4127, 4129-4170, 4173-4195, 4200-4214, 4217-4225, 4228,4230, 4231, 4238, and 4239.

16. A peptide consisting of the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1294-1336, 4002, 4013, 4021, 4023,4025-4031, 4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172,4196-4199, 4215, 4216, 4226, 4277, 4229, 4232, and 4233.

17. A TFPI-binding peptide comprising a homo-dimer or homo-multimer oftwo or more peptides according to any one of paragraphs 1-16.

18. A TFPI-binding peptide comprising a hetero-dimer or hetero-multimerof two or more peptides according to any of the paragraphs 1-16.

19. The peptide according to any one of paragraphs 1-18, wherein thepeptide inhibits TFPI activity and binds to TFPI 1-alpha with adissociation constant of less than 10 μM.

20. The peptide according to any one of paragraphs 1-19, wherein thepeptide is conjugated to a polyethylene glycol (PEG) moiety.

21. The peptide according to any one of paragraphs 1-20, wherein thepeptide is conjugated to human serum albumin (HSA), an antibody orfragment thereof, hydroxyethyl starch, a proline-alanine-serine multimer(PASylation), a C12-C18 fatty acid, or polysialic acid.

22. The peptide according to any one of paragraphs 1-21, wherein thepeptide is conjugated to a moiety selected from the group consisting ofa photosensitizer, dye, a fluorescence dye, a radionuclide, aradionuclide-containing complex, an enzyme, a toxin, an antibody orfragment thereof, and a cytotoxic agent.

23. A peptide according to any one of paragraphs 1-22 for use in amethod for the treatment of a subject.

24. The peptide according to paragraph 24, wherein the method is for thetreatment of a blood coagulation disorder.

25. Use of the peptide according to any one of paragraphs 1-22 for themanufacture of a medicament.

26. Use of the peptide according to any one of paragraphs 1-22 for themanufacture of a medicament for the treatment of a blood coagulationdisorder.

27. A pharmaceutical composition comprising the peptide of any one ofparagraphs 1-22 and a pharmaceutically acceptable carrier.

28. The pharmaceutical composition according to paragraph 28, whereinthe composition comprises a further pharmaceutically effective agent.

29. The pharmaceutical composition according to paragraph 27 orparagraph 28,

wherein the pharmaceutical composition is for use in a method oftreating a blood coagulation disorder.

30. A method for targeting a cell displaying TFPI, the method comprisingcontacting the cell with the peptide of any one of paragraphs 1-22.

31. The method of paragraph 30, wherein the cell is in a mammal, andcontacting the cell comprises administering the peptide to the mammal.

32. The method according to paragraph 30 or paragraph 31 furthercomprising detecting peptide binding to TFPI displayed on the cell.

33. The method according to paragraph 32, wherein peptide-TFPI bindingis detected by detecting a moiety conjugated to the peptide and selectedfrom the group consisting of a photosensitizer, a dye, a fluorescencedye, a radionuclide, a radionuclide-containing complex, an enzyme, atoxin, an antibody, and a cytotoxic agent.

34. The method according to paragraph 32 or paragraph 33, whereinpeptide-TFPI binding is detected by detecting an interaction partnercomplexed with the peptide or a moiety conjugated to the peptide.

35. The method according to paragraph 34, wherein the interactionpartner is selected from the group consisting of an antibody or fragmentthereof, an anticalin, an aptamer, streptavidin, avidin, neutravidin,and a spiegelmer.

36. The method according to paragraph 34 or paragraph 35, wherein theinteraction partner comprises a detection moiety.

37. The method according to paragraph 36, wherein the detection moietyis selected from the group consisting of a dye, a fluorescence dye, aradionuclide, a radionuclide-containing complex, and an enzyme.

38. A method for treating a subject suffering from a disease or being atrisk of suffering from a disease, the method comprising administering tothe subject the peptide of any one of paragraphs 1-22, wherein thepeptide is conjugated to a therapeutic agent.

39. A method for treating a subject suffering from a disease or being atrisk of suffering from a disease, the method comprising administering tothe subject the peptide of any one of paragraphs 1-22, and administeringto the subject an interaction partner that (a) binds the peptide and (b)is a therapeutic agent or is conjugated to a therapeutic agent.

40. The method according to paragraph 39, wherein the therapeutic agentis selected from the group consisting of a photosensitizer, aradionuclide, a radionuclide-containing complex, an enzyme, a toxin, anantibody or fragment thereof, and a cytotoxic agent.

41. The method according to paragraph 39 or paragraph 40, wherein theinteraction partner is selected from the group consisting of an antibodyor fragment thereof, an anticalin, an aptamer, streptavidin, avidin,neutravidin, and a spiegelmer.

42. A method for diagnosing a subject suffering from a disease or beingat risk of suffering from a disease, comprising (a) administering to thesubject the peptide of any one of paragraphs 1-22 conjugated to adetectable moiety and (b) detecting the detectable moiety.

43. A method for diagnosing a subject suffering from a disease or beingat risk of suffering from a disease, comprising (a) administering to thesubject the peptide of any one of paragraphs 1-22, (b) administering tothe subject an interaction partner conjugated to a detectable moiety,and (c) detecting the detectable moiety.

44. The method according to paragraph 43, wherein the interactionpartner is selected from the group consisting of an antibody or fragmentthereof, an anticalin, an aptamer, streptavidin, avidin, neutravidin,and a spiegelmer.

45. The method according to any one of paragraphs 42-44, wherein thedetectable moiety is selected from the group consisting of a dye, afluorescence dye, a radionuclide, a radionuclide-containing complex, anenzyme, and an antibody or fragment thereof.

46. The method according to any one of paragraphs 38-45, wherein thedisease is a blood coagulation disorder.

47. A method for purifying TFPI, wherein the method comprises a)contacting a sample containing TFPI with the peptide of any one ofparagraphs 1-22 under conditions appropriate to form a complex betweenTFPI and the peptide; b) removing the complex from the sample; and,optionally, c) dissociating the complex to release TFPI.

48. The method according to paragraph 47, wherein the peptide isimmobilized to a support.

49. The method according to paragraph 48, wherein the peptide isimmobilized to a chromatography stationary phase, and step (c) compriseseluting TFPI bound to the immobilized peptide.

50. The method according to paragraph 48 or paragraph 49, wherein TFPIis purified via affinity chromatography.

51. A method for identifying a TFPI-binding compound, the methodcomprising (a) contacting a peptide comprising TFPI Kunitz domain 1(KD1) with a TFPI-binding peptide of any one of paragraphs 1-22 and atest compound under conditions that allow formation of KD1-TFPI-bindingpeptide complexes, (b) measuring KD1-TFPI-binding peptide complexesformed in step (a), and (c) comparing the number of KD1-TFPI-bindingpeptide complexes formed in the presence of the test compound with thenumber of KD1-TFPI-binding peptide complexes formed in the absence ofthe test compound, wherein a reduction in the number of KD1-TFPI-bindingpeptide complexes formed in the presence of the test compound comparedto the number of KD1-TFPI-binding peptide complexes formed in theabsence of the test compound indicates that the test compound is aTFPI-binding compound.

52. The method of paragraph 51, wherein the TFPI-binding peptidecomprises a label that generates a signal; step (b) comprises measuringsignal generated by KD1-TFPI-binding peptide complexes; and step (c)comprises comparing signal measured in step (b) with signal generated byKD1-TFPI-binding peptide complexes formed in the absence of the testcompound, wherein a reduction in signal generated by KD1-TFPI-bindingpeptide complexes formed in the presence of the test compound comparedto signal generated by KD1-TFPI-binding peptide complexes formed in theabsence of the test compound indicates that the test compound is aTFPI-binding compound.

53. The method of paragraph 51 or paragraph 52, wherein step (a)comprises (al) contacting the peptide comprising KD1 with theTFPI-binding peptide under conditions that allow formation ofKD1-peptide complexes, and (a2) contacting KD1-TFPI-binding peptidecomplexes formed in step (al) with the test compound.

54. A method for identifying a TFPI-binding compound, the methodcomprising (a) contacting a peptide comprising TFPI Kunitz domain 1(KD1) with a test compound, and (b) detecting binding of the testcompound to a TFPI binding site defined by KD 1 amino acid residuescorresponding to human TFPI residues Phe28, Lys29, Ala30, Asp32, Ile46,Phe47, and Ile55.

55. The method of paragraph 54, wherein the binding site is defined byamino acid residues corresponding to human TFPI residues Ala27, Phe28,Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55.

56. The method of paragraph 54 or paragraph 55, wherein the binding siteis defined by amino acid residues corresponding to human TFPI residuesAla27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44,Ile46, Phe47, and Ile55.

57. The method of any one of paragraphs 54-56, wherein step (b)comprises determining the presence or absence of a nuclear magneticresonance (NMR) chemical shift within the TFPI binding site.

58. The method of any one of paragraphs 54-56, wherein step (a)comprises contacting the peptide comprising TFPI KD1 with FVIIa in thepresence of a test compound under conditions that allow binding of KD1to FVIIa, and step (b) comprises comparing KD1-FVIIa binding in step (a)with KD1-FVIIa binding in the absence of the test compound, wherein adecrease in KD1-FVIIa binding in the presence of the test compoundcompared to KD1-FVIIa binding in the absence of the test compoundindicates that the test compound is a TFPI-binding compound.

59. The method of any one of paragraphs 54-56, wherein step (a)comprises contacting the peptide comprising TFPI KD1 with FXa in thepresence of a test compound under conditions that allow binding of KD1to FXa, and step (b) comprises comparing KD1-FXa binding in step (a)with KD1-FXa binding in the absence of the test compound, wherein adecrease in KD1-FXa binding in the presence of the test compoundcompared to KD1-FXa binding in the absence of the test compoundindicates that the test compound is a TFPI-binding compound.

60. The method of any one of paragraphs 54-56, wherein the peptidecomprising TFPI KD1 further comprises Kunitz domain 2 (KD2), step (a)comprises contacting the peptide comprising TFPI KD1 and TFPI KD2 withFXa in the presence of a test compound under conditions that allowbinding of KD2 to FXa, and step (b) comprises comparing KD2-FXa bindingin step (a) with KD2-FXa binding in the absence of the test compound,wherein a decrease in KD2-FXa binding in the presence of the testcompound compared to KD2-FXa binding in the absence of the test compoundindicates that the test compound is a TFPI-binding compound.

61. The method of any one of paragraphs 54-56 and 58-60, wherein bindingof the test compound to the TFPI binding site is detected using anenzymatic assay.

62. The method of any one of paragraphs 54-61, wherein the peptidecomprising TFPI KD1 comprises amino acids 1-160 of human TFPI.

63. The method of any one of paragraphs 54-61, wherein the peptidecomprising TFPI KD1 is full length human TFPI.

64. A composition comprising a TFPI inhibitor identified by the methodof any one of paragraphs 51-63.

65. Use of a TFPI inhibitor identified by the method of any one ofparagraphs 51-63 for the manufacture of a medicament.

66. Use of a TFPI inhibitor identified by the method of any one ofparagraphs 51-63 for the manufacture of a medicament for treating ablood coagulation disorder.

67. A method for treating a subject suffering from a disease or being atrisk of suffering from a disease, the method comprising administering tothe subject a TFPI inhibitor identified by the method of any one ofparagraphs 51-63.

68. A method for inhibiting human TFPI, the method comprising contactinghuman TFPI with an inhibitor that binds human TFPI at a binding sitedefined by amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47,and Ile55.

69. A method for treating a subject suffering from a disease or at riskof suffering from a disease, the method comprising administering to thesubject an inhibitor that binds human TFPI at a binding site defined byamino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55.

70. The method of paragraph 68 or paragraph 69, wherein the human TFPIbinding site is defined by amino acid residues Ala27, Phe28, Lys29,Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55.

71. The method of paragraph 70, wherein the human TFPI binding site isdefined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32,Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55.

72. A method for purifying a compound that inhibits FXa activity, themethod comprising (a) contacting a peptide comprising TFPI Kunitz domain1 (KD1) with a compound under conditions that allow formation ofcompound-KD1 complexes, (b) removing unbound compound, and (c)dissociating the compound-KD1 complexes to release the compound.

73. The method of paragraph 72, wherein step (a) comprises contactingthe peptide comprising KD1 with a population of test compounds.

74. A computer storage media having computer executable instructionsthat, when executed on the processor of a computer, implement a methodof modeling interaction between selected three dimensional (3D) pointsin a TFPI Kunitz domain 1 (KD1) protein and a test compound, the methodcomprising: obtaining a protein structure 3D model for the TFPI KD1protein; determining a 3D relationship between a selected subset ofamino acids in the protein structure, wherein the selected subset ofamino acids comprises Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, andIle55; modeling a surface bounded by the selected subset of amino acids;obtaining a test compound 3D model of a test compound; matching the testcompound 3D model to the surface bounded by the selected subset of aminoacids; and identifying contact points between the selected subset ofamino acids of the surface and the test compound 3D model.

75. The computer storage media of paragraph 74, wherein the selectedsubset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp31,Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55.

76. The computer storage media of paragraph 74, wherein the selectedsubset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp31,Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55.

77. The computer storage media of any one of paragraphs 74-76, furthercomprising: determining a number of the contact points between thesurface and the test compound 3D model; and recording an affinity ratingfor the test compound 3D model corresponding to the number of contactpoints.

78. The computer storage media of any one of paragraphs 74-77, whereinthe test compound is a peptide.

79. The computer storage media of any one of paragraphs 74-78, furthercomprising: determining a bond type for each contact point between thesurface and the test compound 3D model; and updating the affinity ratingbased on an aggregate of the bond types for each contact point betweenthe surface and the test compound 3D model.

80. The computer storage media of any one of paragraphs 74-79, furthercomprising: obtaining an updated test compound 3D model based on asecond test compound; matching the updated test compound 3D model to thesurface bounded by the selected subset of amino acids; and identifyingthe identified contact points between the selected subset of amino acidsof the surface and the updated test compound 3D model on a display ofthe computer.

81. The computer storage media of paragraph 80, further comprising:determining a number of the contact points between the surface and theupdated test compound 3D model; determining a bond type for each contactpoint between the surface and the updated test compound 3D model; andrecording a new affinity rating based on the number of contact pointsand an aggregate of the bond types for each contact point between thesurface and the updated test compound 3D model.

82. The computer storage media of paragraph 81, further comprising:comparing the updated affinity rating with the new affinity rating todetermine whether the test compound or the second test compound has ahigher affinity rating.

83. The computer storage media of any one of paragraphs 80-82, whereinthe second test compound is a variant of the test compound.

84. The computer storage media of any one of paragraphs 74-83, furthercomprising displaying the contact points on a display of the computer.

85. The computer storage media of any one of paragraphs 78-84, furthercomprising modifying the peptide to increase the number of contactpoints with the selected subset of amino acids or increase bond strengthbetween amino acids of the peptide and the selected subset of aminoacids.

86. A method of comparing a test compound to selected three dimensionalpoints in a TFPI Kunitz domain 1 (KD1) protein, the method comprising:creating a protein structure for the KD1 protein in a memory of acomputer; determining a three dimensional model of a selected subset ofamino acids in the KD1 protein at a processor of the computer, whereinthe selected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32,Ile46, Phe47, and Ile55; determining a three dimensional model of a testcompound at the processor of the computer; fitting the 3D model of thetest compound to the 3D model of the selected subset of amino acids atthe processor of the computer; and generating an affinity of the testcompound for the selected subset of amino acids at the processor of thecomputer, wherein the affinity is based on a number of amino acids inthe subset in contact with the test compound and a bond strength at eachcontact point.

87. The method of paragraph 86, wherein the selected subset of aminoacids comprises Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ile38,Ile46, Phe47, and Ile55.

88. The method of paragraph 86, wherein the selected subset of aminoacids comprises Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37,Ile38, Phe44, Ile46, Phe47, and Ile55.

89. The method of any one of paragraphs 86-88, further comprising:displaying a 3D representation of the fit between the test compound andthe 3D model of the selected subset of amino acids.

90. The method of any one of paragraphs 86-89, further comprising:repeating the steps of paragraph 86 for a plurality of test compounds;and saving the respective affinities for each of the plurality of testcompounds.

91. A computer storage media having computer executable instructionsthat, when executed on the processor of a computer, implement a methodof comparing a peptide to selected three dimensional points (3D) in aTFPI Kunitz domain 1 protein (KD1), the method comprising: creating aprotein structure for the KD1 protein; determining a three dimensionalmodel of a selected subset of amino acids in the KD1 protein, whereinthe subset of amino acids comprises Phe28, Lys29, Ala30, Asp32, Ile46,Phe47 and Ile55; determining a three dimensional model of a peptide;fitting the 3D model of the peptide to the 3D model of the selectedsubset of amino acids; and generating an affinity of the peptide for theselected subset of amino acids, wherein the affinity is based on anumber of amino acids in the subset in contact with the peptide and abond strength at each contact point.

92. The computer storage media of paragraph 91, wherein the selectedsubset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp31,Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55.

93. The computer storage media of paragraph 91, wherein the selectedsubset of amino acids comprises Ala27, Phe28, Lys29, Ala30, Asp31,Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55.

DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of the blood coagulation cascade.

FIG. 2 is an illustration of the secondary structure of Tissue FactorPathway Inhibitor-1.

FIG. 3 is an illustration of the formation of a quaternary complexcomprising Tissue Factor, Factor Xa (FXa), Factor VIIa (FVIIa), andTFPI.

FIG. 4 is a listing of amino acid sequences of various TFPI-inhibitorypeptides denoting amino acid substitutions (bolded and underlined) inreference to peptide JBT0293.

FIG. 5 is an illustration of mRNA display selection of TFPI-inhibitorpeptides.

FIG. 6A is an illustration of the EC₅₀ binding ELISA and FIG. 6B is anillustration of the IC₅₀ ELISA described in Example 1.

FIG. 7 is a binding ELISA curve comparing % OD (y-axis) andconcentration [nM] (x-axis) for biotinylated peptide JBT0132.

FIGS. 8A-8D are competition ELISA curves comparing % OD (y-axis) andconcentration [nM] (x-axis) for exemplary peptides of the invention.

FIGS. 9A and 9B are sensorgrams plotting RU (y-axis) against time inseconds (x-axis) for peptides JBT0120 and JBT0132.

FIGS. 10A and 10B are sensorgrams plotting RU (y-axis) against time inseconds (x-axis) for peptide JBT0120 interaction with Tissue FactorPathway Inhibitor-1 and Tissue Factor Pathway Inhibitor-2.

FIGS. 11A and 11B are graphs comparing amount of thrombin generated (nM)(y-axis) and time in minutes (x-axis) for peptide JBT0120 and peptideJBT0132 in a plasma-based assay.

FIGS. 12A-12P, 13A-13D, 14A-14E, 15A-15D, 16A-16F, 17A-17B, and 18A-18Eare tables listing the amino acid sequences of various TFPI-inhibitorypeptides; EC₅₀ and percent inhibition of TFPI observed in the FXainhibition assay; EC₅₀ and percent inhibition of TFPI observed in theextrinsic tenase inhibition assay; and FEIBA, Factor VIII (FVIII)Immunate, or Factor IX (FIX) equivalent activities (mU/mL) inplasma-based assays. “*” denotes negative controls.

FIGS. 19A-19B, and 20-21 are tables listing the results from BIAcoreanalysis of several TFPI-binding peptides. “*” denotes negativecontrols.

FIGS. 22A-22M, 23A-23L, 24A-24D, 25A-25L, 26A-26F, 27A-27C, 28A-28N,29A-29F, and 30A-30D are tables listing the amino acid sequences ofvarious TFPI-binding peptides; EC₅₀ and percent inhibition of TFPIobserved in the FXa inhibition assay; EC₅₀ and percent inhibition ofTFPI observed in the extrinsic tenase inhibition assay; and FEIBA, FVIIIImmunate, or FIX equivalent activities (mU/mL) in plasma-based assays.“*” denotes negative controls.

FIG. 31 is a graph comparing a pharmacokinetic characteristic(concentration of peptide (y-axis) versus time after administration(x-axis)) of a PEGylated TFPI-binding peptide to the pharmacokineticcharacteristic of same peptide lacking PEG. The peptides wereadministered intravenously to C57B16 mice at a dose of 10 mg/kg. Threebiological samples were analyzed for the presence of peptide at eachtime point.

FIGS. 32A-32AM, 33, 34A-34J, 35, 36A-36Q, 37, 38A-38B, and 39 are tableslisting the amino acid sequences and IC₅₀ or EC₅₀ values of variouspeptides of the invention. “*” denotes negative controls.

FIG. 40 is a graph illustrating a pharmacokinetic characteristic(concentration of peptide (nM) (y-axis) versus time after administration(minutes) (x-axis)) of a PEGylated TFPI-binding peptide followingsubcutaneous administration to mice at a dose of 10 mg/kg.

FIG. 41 is a graph correlating the amount of thrombin generated (nM)(y-axis) with time (minutes) (x-axis) for peptide JBT1855 in aplasma-based assay of hemophilia A patient plasma.

FIG. 42 is a graph illustrating the amount of blood loss (μl; y-axis)observed following a nail-clip in mice treated with JBT-1855(intravenous or subcutaneous administration), anti-TFPI antibody(intravenous administration), or vehicle (intravenous administration)(x-axis).

FIG. 43 is a graph plotting TFPI160 amino acid residue (x-axis) againstthe chemical shift differences of HSQC signals for free TFPI160 andTFPI160 bound to JBT0303 (y-axis).

FIG. 44 is a ribbon model of the secondary structure of TFPIillustrating regions of chemical shift changes of HSQC signals whenTFPI160 is complexed to JBT0303 compared to uncomplexed (free) TFPI160.

FIG. 45 is a graph plotting TFPI160 amino acid residue (x-axis) againstthe chemical shift differences of HSQC signals for free TFPI160 andTFPI160 bound to JBT0122 (y-axis).

FIG. 46 is a ribbon model of the secondary structure of TFPI proteinillustrating regions of chemical shift changes of HSQC signals whenTFPI160 is complexed to JBT0122 compared to uncomplexed (free) TFPI160.

FIG. 47 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0788 based on HSQC, HNCACB, HNCA, HNCO and HNNspectra.

FIG. 48 is a ribbon model of the secondary structure of free JBT0788.

FIG. 49 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0788 complexed with TFPI160 based on HSQC,HNCACB, HNCA, HCCOCA, and HNCO spectra.

FIG. 50 is a ribbon model of the secondary structure of JBT0788 whencomplexed with TFPI160.

FIG. 51 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0616 based on HSQC, HNCACB, and HNN spectra.

FIG. 52 is a ribbon model of the secondary structure of free JBT0616.

FIG. 53 is a table listing assignments for the carbonyl carbon (C), thealpha carbon (CA), the beta carbon (CB), the amide proton (H), and theamide nitrogen (N) of JBT0616 complexed with TFPI based on HSQC, HNCO,HNCA, and HNCOCA spectra.

FIG. 54 is a ribbon model of the secondary structure of JBT0616 whencomplexed with TFPI160.

FIG. 55 is a ribbon structure of the energetically minimized best modelof KD1 (residues 22-79) in complex with JBT0303 with residues proposedto drive the protein-protein interaction displayed as sticks. Italicizedand underlined residues belong to JBT0303; the remaining residues belongto KD1 of TFPI.

FIG. 56 is a rotational thromboelastogram correlating sample elasticity(mm) with time in seconds (s) for JBT2317.

FIG. 57 is a rotational thromboelastogram correlating sample elasticity(mm) with time in seconds (s) for JBT2329.

FIG. 58 is an illustration of a computing device.

FIG. 59 is an illustration of a three dimensional (3D) model of a KD1protein.

FIG. 60 is an illustration of a 3D model of a TFPI-binding peptide.

FIG. 61 is an illustration of a method of modeling protein and peptideinteraction.

FIG. 62A-62F is a table listing the amino acid sequences and IC₅₀ orEC₅₀ values of various peptides of the invention. Designation “n.a.” is“not analyzed.” Progression curve data were obtained using the FXainhibition assay described in Example 3 with recombinant human fulllength TFPI. Assay concentration of progression curve assay was 0.0025%(0.1% Tween80 used in peptide dilution buffer).

FIG. 63 is a graph correlating concentration of peptides JBT2325-JBT2329(nM) (y-axis) with time following intravenous administration (hours)(x-axis). Peptides comprising higher weight PEG moieties exhibited aprolonged in vivo half life in mice. Each time point is represented bythe mean of three independent samples quantified by ELISA.

FIG. 64A-64C are graphs correlating concentration of peptides JBT2401,JBT2404 and JBT2410 (nM) (y-axis) with time following intravenousadministration (hours) (x-axis). Each time point is represented by themean of three independent samples quantified by ELISA. Solid circlessymbolize intravenous data, solid triangles symbolize subcutaneous data.

FIG. 65A-65B is a table listing the amino acid sequences of variouspeptides of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides peptides that bind Tissue Factor PathwayInhibitor-1 and, in some instances, block the inhibitory activity ofTissue Factor Pathway Inhibitor-1 (herein referred to as TFPI) withinthe blood coagulation cascade. Upon vascular injury, Tissue Factor (TF)complexes with Factor Vlla to form the “extrinsic complex” or “extrinsictenase complex,” which activates Factors IX and X (FIG. 1). TFPI is themain natural regulator of TF/FVIIa extrinsic complex activity and byextension, plays a role in controlling thrombin generation (Panteleev etal., Eur. J. Biochem., 249, 2016-2031 (2002)). TFPI is a 43 kDa serineprotease inhibitor comprising three Kunitz-type inhibitory domains (FIG.2). Kunitz domain 1 of TFPI binds FVIIa and Kunitz domain 2 binds FXa,enabling the inhibitor to form a quaternary FXa-TFPI-FVIIa-TF complexthat blocks activity of the TF/FVIIa extrinsic complex (FIG. 3). TFPIbinding of FXa also downregulates the common pathway of the coagulationcascade, during which FXa converts prothrombin to thrombin (Audu et al.,Anesth. Analg., 103(4), 841-845 (2006)). The invention provides, e.g.,TFPI-inhibitory peptides that block TFPI's inhibitory action on theblood coagulation cascade, thereby enhancing thrombin formation.

The amino acid sequences of several TFPI-binding peptides are providedherein. Conventional amino acids are identified according to theirstandard, one-letter or three-letter codes, as set forth in Table 1.

TABLE 1 3-letter 1-letter codes code Amino acids Ala A Alanine Cys CCysteine Asp D Aspartic acid Glu E Glutamic acid Phe F Phenylalanine GlyG Glycine His H Histidine Ile I Isoleucine Lys K Lysine Leu L LeucineMet M Methionine Asn N Asparagine Pro P Proline Gln Q Glutamine Arg RArginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan TyrY Tyrosine

Examples of non-conventional amino acids and additional peptide buildingblocks are identified according to a three-letter code (with theexception of Ttds and Dopa, which are common four-letter abbreviations)found in Table 2. Additional building blocks designated by three-, four-or seven-number/letter designations or abbreviations also are listed inTable 2. The structures of some building blocks are depicted with anexemplary reagent for introducing the building block into a peptide(e.g., the structure provided for 2-naphthyl sulfonyl comprises achloride).

TABLE 2 Name Abbreviation Structure Phenyl acetyl  374

2-Naphthyl sulfonyl  972

1-Naphthyl sulfonyl  973

3-Phenyl propionyl 1281

Hexanoyl 1525

3-Methyl butanoyl 3067

2-Methyl propionyl 4635

2-(Naphth-2-yl) acetyl 5963

N-(4-aminobutyl)-glycine Abg

2-aminobutyric acid Abu

2-Amino-isobutyric acid Aib

2-Aminoindane-2-carboxylic acid Aic

L-alpha-Methyl leucine Aml

1-Amino-(4-N- piperidinyl)carboxylic acid Apc

N-(4-aminopropyl)-glycine Apg

D-Azetidine-2-carboxylic acid aze

β-Alanine Bal

β-Homoglutamatic acid Bhe

β-Homophenylalanine Bhf

β-Homolysine Bhk

β-Homoleucine Bhl

β-Homoasparagine Bhn

β-Homoglutamine Bhq

β-Homoarginine Bhr

β-Homoserine Bhs

β-Homotyrosine Bhy

β-Homoaspartic acid Bhd

β-Homovaline Bhv, Btl

β-Homoasparagin Bhn, Btq

L-3-Benzothienylalanine Bta

3-(Acetylamino- methylsulfanyl)-2-amino- propionic acid C(Acm)

Aminoethylthiol Cea

(S)-Cyclohexylalanine Cha

L-Cyclohexylglycine Chg

(S)-Citrullin Cit

Carboxymethylen cystein Cmc

N-ethylmaleiimido cysteine C(NEM)

L-Cyclopentylglycine Cpg

(S)-2,4-Diaminobutyric acid Dab

(S)-Diaminopropionic acid Dap

alpha,alpha-Diethylglycine Deg

5,5-Dimethyl-D-thiazolidine-4- carboxylic acid dtc

3,4-Dihydroxyphenylalanine Dopa

(S)-2-Propargylglycine Eag

1-Amino-cyclopropane-1- carboxylic acid Ebc

1-Amino-cyclopentane-1- carboxylic acid Eca

Cys(3-propionic acid amide) Ecl

Sulfoxid of Carboxyethylcystein Ede(O)

Cys(5-methylen-2-oxazolidinon) Eea

Cys(1-methylen-1H- benzotriazol) Eec

Cys(3-methylen-2- benzothiazolinon) Eef

(S)-N(omega)-nitro-arginine Eew

alpha,alpha-Dibutylglycine Egt

1-amino-cyclohexane-1- carboxylic acid Egz

L-homophenylalanine Hfe

(S)-Homo-arginine Har

(S)-Homo-citrulline Hci

(S)-Homo-cysteine Hcy

D-Homo-cysteine hcy

(S)-2-Amino-5-methyl-hexanoic acid Hle

(S)-Homo-lysine Hly

2-Amino-6-(2-aminooxy- acetylamino)-hexanoic acid K(AOA)

1-Naphthylalanine 1Ni

2-Naphthylalanine 2Ni

N-(cyclohexyl)-glycine Ncg

4-Nitrophenyl alanine Nif

(S)-Norleucine Nle

(S)-N-Methylalanine Nma

(S)-N-Methyl-Aspartic acid Nmd

(S)-N-Methyl-glutamic acid Nme

(S)-N-Methyl-phenylalanine Nmf

N-Methyl-glycine Nmg

(S)-N-Methyl-lysine Nmk

(S)-N-Methyl-leucine Nml

N-Methyl-asparagine Nmn

(S)-N-Methyl-arginine Nmr

(S)-N-Methyl-serine Nms

(S)-N-Methyl-valine Nmv

(S)-N-Methyl-tyrosine Nmy

N-propyl glycine NpropylG

(S)-2-Amino-pentanoic acid Nva

(S)-2-Pyridyl-alanine Opa

Ornithine-(pyrazin-carboxylate) Opc

D-Octahydroindol-2-carboxylic acid oic

(S)-Ornithine Orn

Palmitoyl Palm

L-Phenylglycin Phg

4-Phenyl-butyric acid PhPrCO

Polyethylene glycol PEG D-Pipecolic acid pip

L-Tyrosin(O-Methyl)-OH Pmy

L-Phosphotyrosine Pty

N-Methylglycine Sar

Selenomethionine Sem

L-2-Thienylalanine Thi

D-thiazolidine-4-carboxylic acid thz

1,2,3,4-L- tetrahydroisoquinolinecarboxilic acid Tic

L-alpha-t-Butylglycine Tle

(13-Amino-4,7,10-trioxa- tridecayl)-succinamic acid Ttds

Ttds- Maleimidopropionyl(EtSH))

3-Nitro-L-tyrosine Tym

Carboxyfluorescein FAM

[2-(2-Amino-ethoxy)-ethoxy]- acetic acid FA03202

3-{2-[2-(2-Amino-ethoxy)- ethoxy]-ethoxy}-propionic acid FA19203

3-(2-{2-[2-(2-Amino-ethoxy)- ethoxy]-ethoxy}-ethoxy)- propionic acidFA19204

3-[2-(2-{2-[2-(2-Amino- ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionic acid FA19205

The amino acid sequences of the peptides provided herein are depicted intypical peptide sequence format, as would be understood by the ordinaryskilled artisan. For example, the three-letter code or one-letter codeof a conventional amino acid, or the three-, four-, orseven-number/letter code additional building blocks, indicates thepresence of the amino acid or building block in a specified positionwithin the peptide sequence. The code for each non-conventional aminoacid or building block is connected to the code for the next and/orprevious amino acid or building block in the sequence by a hyphen.Adjacent amino acids are connected by a chemical bond (typically anamide bond). The formation of the chemical bond removes a hydroxyl groupfrom the 1-carboxyl group of the amino acid when it is located to theleft of the adjacent amino acid (e.g., Hle-adjacent amino acid), andremoves a hydrogen from the amino group of the amino acid when it islocated on the right of the adjacent amino acid (e.g., adjacent aminoacid-Hle). It is understood that both modifications can apply to thesame amino acid and apply to adjacent conventional amino acids presentin amino acid sequences without hyphens explicitly illustrated. Where anamino acid contains more than one amino and/or carboxy group in theamino acid side chain, the 2- or 3-amino group and/or the 1-carboxygroup generally are used for the formation of peptide bonds. Fornon-conventional amino acids, a 3-letter code was used where the firstletter indicates the stereochemistry of the C-α-atom. For example, acapital first letter indicates that the L-form of the amino acid ispresent in the peptide sequence, while a lower case first letterindicating that the D-form of the correspondent amino acid is present inthe peptide sequence. When one-letter code is used, a lower case letterrepresents a D-amino acid, while an upper case letter represents anL-amino acid. Unless indicated to the contrary, the amino acid sequencesare presented herein in N- to C-terminus direction.

The C-termini of several TFPI-binding peptide sequences described hereinare explicitly illustrated by inclusion of an OH, NH₂, or anabbreviation for a specific terminating amine linked to the C-terminalamino acid code via a hyphen. The N-termini of several peptidesdescribed herein are explicitly illustrated by inclusion of a hydrogen(for a free N-terminus), or an abbreviation for a specific terminatingcarboxylic acid or other chemical group linked to the N-terminal aminoacid code via a hyphen.

The invention provides a peptide comprising the amino acid sequenceX₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁ (SEQ ID NO: 3109), wherein(using single letter codes for amino acids)

X₇ is selected from the group consisting of L, P, K, S, W, V, N, and Q;

X₈ is selected from the group consisting of L, R, N, F, and I;

X₉ is selected from the group consisting of Y, V, P, and C;

X₁₀ is selected from the group consisting of F, L, and G;

X₁₁ is selected from the group consisting of L, W, V, A, M, T, and S;

X₁₂ is selected from the group consisting of T, F, V, R, A, D, L, E, S,and Y;

X₁₃ is selected from the group consisting of I, M, G, Q, D, and R;

X₁₄ is selected from the group consisting of G, W, Y, L, M, and H;

X₁₅ is selected from the group consisting of N, P, F, H, K, and Y;

X₁₆ is selected from the group consisting of M, D, E, V, G, and K;

X₁₇ is selected from the group consisting of G, I, R, S, T, and L;

X₁₈ is selected from the group consisting of M, K, L, and I;

X₁₉ is selected from the group consisting of Y, G, R, and S;

X₂₀ is selected from the group consisting of A, E, S, C, and Y; and

X₂₁ is selected from the group consisting of A, V, K, and E.

In addition to the core structure set forth above, X₇-X₂₁, otherstructures that are specifically contemplated are those in which one ormore additional amino acids are attached to the core structure (e.g.,linked to the N-terminus or the C-terminus of the amino acid sequenceX₇-X₂₁). Thus, the invention includes peptides comprising the corestructure and further comprising one or more N-terminal amino acid(s)comprising an amino acid sequence selected from the group consisting of:

X₆,

X₅X₆,

X₄X₅X₆,

X₃X₄X₅X₆ (SEQ ID NO: 3110),

X₂X₃X₄X₅X₆ (SEQ ID NO: 3111), and

X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 3112);

wherein X₆ is directly linked to X₇ of the core structure amino acidsequence, and

X₁ is selected from the group consisting of T and G;

X₂ is selected from the group consisting of F and V;

X₃ is selected from the group consisting of V, W, Y, and F;

X₄ is selected from the group consisting of D, Q, and S;

X₅ is selected from the group consisting of E, T, N, and S; and

X₆ is selected from the group consisting of R, H, K, and A.

The peptide of the invention in one aspect comprises or consists of theamino acid sequence QSKKNVFVFGYFERLRAK (SEQ ID NO: 1).

In another embodiment, the peptide of the invention comprising the corestructure comprises one or more C-terminal amino acid(s) comprising anamino acid sequence selected from the group consisting of:

X₂₂,

X₂₂X₂₃,

X₂₂X₂₃X₂₄,

X₂₂X₂₃X₂₄X₂₅ (SEQ ID NO: 3113),

X₂₂X₂₃X₂₄X₂₅X₂₆ (SEQ ID NO: 3114), and

X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇ (SEQ ID NO: 3115),

wherein X₂₂ is directly linked to X₂₁ of the core structure amino acidsequence, and

X₂₂ is selected from the group consisting of Q, I, E, W, R, L, and N;

X₂₃ is selected from the group consisting of L, V, M, and R;

X₂₄ is selected from the group consisting of K, L, A, and Y;

X₂₅ is F;

X₂₆ is G; and

X₂₇ is T.

In one aspect, the peptide of the invention comprises or consists of theamino acid sequence VIVFTFRHNKLIGYERRY (SEQ ID NO: 4). It is alsocontemplated that the peptide of the invention comprises additionalamino acids at both the N-terminus and the C-terminus of the corestructure. In this aspect, the peptide comprises or consists of theamino acid sequence TFVDERLLYFLTIGNMGMYAAQLKF (SEQ ID NO: 3),GVWQTHPRYFWTMWPDIKGEVIVLFGT (SEQ ID NO: 5), KWFCGMRDMKGTMSCVWVKF (SEQ IDNO: 6), or ASFPLAVQLHVSKRSKEMA (SEQ ID NO: 7).

The invention further includes peptides comprising the amino acidsequence X₃X₄X₅-F-X₇-NVF-X₁₁X₁₂-GY-X₁₅X₁₆-RLRAK-X₂₂ (SEQ ID NO: 2),wherein X₃ is Y or F; X₄ is Q or S; X₅ is N or S; X₇ is K, N, or Q; X₁₁is V, A, S, or T; X₁₂ is F, A, D, L, Q, S, or Y; X₁₅ is F, K, or Y; X₁₆is E or D; and X₂₂ is L or N.

In addition, the invention provides a peptide that binds TFPI, whereinthe peptide comprises the structure of formula (I):X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019-X1020(SEQ ID NO: 3116). In formula (I),

X1001 is an amino acid selected from the group consisting of Bhf, C, D,F, G, H, I, K, L, M, N, Nmf, Q, R, T, V, W, and Y;

X1002 is an amino acid selected from the group consisting of G, K, andQ;

X1003 is an amino acid selected from the group consisting of A, Aib,Bhs, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

X1004 is an amino acid selected from the group consisting of, A, Aib,Bhk, C, D, E, F, G, H, I, K, k, L, M, N, Nmk, P, Q, R, S, T, V, W, andY;

X1005 is an amino acid selected from the group consisting of a, A, Aib,Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T, V, W, andY;

X1006 is an amino acid selected from the group consisting of A, Aib,Btq, C, D, E, F, G, H, I, K, L, M, N, Q, R, S T, V, W, and Y;

X1007 is an amino acid selected from the group consisting of A, F, G, I,K, L, Nmv, P, Q, S, V, W, and Y;

X1008 is an amino acid selected from the group consisting of F, H, K, W,and Y;

X1009 is an amino acid selected from the group consisting of A, Aib, f,I, K, S, T, and V;

X1010 is an amino acid selected from the group consisting of A, Aib, C,D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, and Y;

X1011 is an amino acid selected from the group consisting of Aib, C, K,G, and Nmg;

X1012 is Y;

X1013 is an amino acid selected from the group consisting of A, Aib, C,E, F, G, H, K, L, M, Q, R, W, and Y;

X1014 is an amino acid selected from the group consisting of A, Aib,Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

X1015 is an amino acid selected from the group consisting of(omega-methyl)-R, D, E, K, and R;

X1016 is L;

X1017 is an amino acid selected from the group consisting of(omega-methyl)-R, A, Aib, Bhr, C, Cha, Cit, D, Dab, Dap, E, Eag, Eew, F,G, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, Q, R, S, T, V,W, and Y;

X1018 is an amino acid selected from the group consisting of A, Bal, C,D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; and

X1019 is an amino acid selected from the group consisting of Bhk, K, R,and V.

X1020 is either present or absent in formula (I) (i.e., in someinstances, the peptide of the invention comprises the structureX1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019(SEQ ID NO: 3116)). When X1020 is present, it is an amino acid selectedfrom the group consisting of Aib, Bhl, C, F, G, H, I, K, L, Nml, Q, R,S, T, V, W, and Y.

For example, the peptide of the invention comprises the structure offormula (I) wherein X1001 is an amino acid selected from the groupconsisting of C, F, I, K, L, Nmf, V, M, W, and Y; X1002 is Q; X1003 isan amino acid selected from the group consisting of A, C, D, E, H, K, M,I, N, Q, R, S, T, and V; X1004 is an amino acid selected from the groupconsisting of A, Aib, C, D, E, G, H, F, I, K, k, L, M, N, Nmk, P, Q, R,S, V, W, and Y; X1005 is an amino acid selected from the groupconsisting of a, A, Aib, Bal, C, d, E, D, F, G, H, K, k, L, M, N, Nmg,p, Q, R, S, T, and Y; X1006 is an amino acid selected from the groupconsisting of A, Btq, C, D, G, I, K, H, L, M, N, Q, R, S, V, and Y;X1007 is an amino acid selected from the group consisting of I, K, L, Q,V, and Y; X1008 is an amino acid selected from the group consisting ofF, H, and Y; X1009 is an amino acid selected from the group consistingof f, I, and V; X1010 is an amino acid selected from the groupconsisting of A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, and Y;X1011 is an amino acid selected from the group consisting of G and Nmg;X1012 is Y; X1013 is an amino acid selected from the group consisting ofAib, C, F, H, L, W, and Y; X1014 is an amino acid selected from thegroup consisting of A, Aib, Bhe, C, D, E, H, I, K, L, M, N, Q, R, S, T,V, W, and Y; X1015 is an amino acid selected from the group consistingof E and R; X1016 is L; X1017 is an amino acid selected from the groupconsisting of (omega-methyl)-R, A, Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag,Eew, F, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, R, S, T, V,and Y; X1018 is an amino acid selected from the group consisting of A,C, D, E, F, I, K, L, M, N, Q, R, V, and W; X1019 is an amino acidselected from the group consisting of K and R; and X1020 is an aminoacid selected from the group consisting of Aib, Bhl, F, K, L, R, and W(when X1020 is present in the peptide).

In one aspect, the peptide of the invention comprises the structure offormula (I) wherein X1001 is an amino acid selected from the groupconsisting of F, L, Y, and M; X1002 is Q; X1003 is an amino acidselected from the group consisting of M, Q, R, S, T, and C; X1004 is anamino acid selected from the group consisting of Aib, K, L, P, R, E, G,I, Y, M, and W; X1005 is an amino acid selected from the groupconsisting of a, Aib, D, d, G, H, K, k, N, Nmg, p, Q, R, A, E, C, and M;X1006 is an amino acid selected from the group consisting of A, C, D, G,H, K, N, Q, R, S, and M; X1007 is an amino acid selected from the groupconsisting of I and V; X1008 is an amino acid selected from the groupconsisting of F, H, and Y; X1009 is V; X1010 is an amino acid selectedfrom the group consisting of A, D, E, K, M, N, Q, R, F, H, P, S, V, W,and Y; X1011 is G; X1012 is Y; X1013 is C or F; X1014 is an amino acidselected from the group consisting of A, C, D, E, K, L, M, N, Q, R, T,V, and Aib; X1015 is R; X1016 is L; X1017 is an amino acid selected fromthe group consisting of A, Aib, C, Cha, Dab, Dap, Eag, Eew, H, Har, Hci,Hle, K, Nle, Nva, Opa, Orn, R, I, L, S, and M; X1018 is an amino acidselected from the group consisting of A, L, N, M, and R; X1019 is K; andX1020 is K or L.

When amino acid X1020 is absent from formula (I), the peptide of theinvention in one aspect further comprises amino acid X1000 at theN-terminus of formula (I), such that the peptide comprises or consistsof the structure of formula (II):X1000-X1001-X1002-X1003-X1004-X1005-X1006-X1007-X1008-X1009-X1010-X1011-X1012-X1013-X1014-X1015-X1016-X1017-X1018-X1019(II) (SEQ ID NO: 3122). When X1000 is present in the peptide, X1000 isan amino acid selected from the group consisting of A, E, and P, whilethe amino acids of X1001-X1019 are as defined above.

In an additional aspect, the TFPI-binding peptide of the inventioncomprises the structure of formula (III):X1001-Q-X1003-X1004-X1005-X1006-I/V-X1008-V-X1010-G-Y-C/F-X1014-R-L-X1017-X1018-K-K/L(III) (SEQ ID NO: 3117). As used herein, amino acid designationsseparated by “/” refer to alternative amino acid residues at theindicated position. For example, with respect to formula (III), theamino acid residue at position 7 is isoleucine or valine. X1001, X1003,X1004, X1005, X1006, X1008, X1010, X1014, X1017 and X1018 in formula(III) are each independently selected from any amino acid. For example,in formula (III),

X1001 is optionally an amino acid selected from the group consisting ofBhf, C, D, F, G, H, I, K, L, M, N, Nmf, Q, R, T, V, W, and Y, such as anamino acid selected from the group consisting of C, F, I, K, L, Nmf, V,M, W, and Y (e.g., an amino acid selected from the group consisting ofF, L, Y and M);

X1003 is optionally an amino acid selected from the group consisting ofA, Aib, Bhs, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, andY, such as an amino acid selected from the group consisting of A, C, D,E, H, K, M, I, N, Q, R, S, T, and V (e.g., the amino acid is M, Q, R, S,T or C);

X1004 is optionally an amino acid selected from the group consisting of,A, Aib, Bhk, C, D, E, F, G, H, I, K, k, L, M, N, Nmk, P, Q, R, S, T, V,W, and Y, such as an amino acid selected from the group consisting of A,Aib, C, D, E, G, H, F, I, K, k, L, M, N, Nmk, P, Q, R, S, V, W, and Y(e.g., an amino acid selected from the group consisting of Aib, K, L, P,R, E, G, I, Y, M, and W);

X1005 is optionally an amino acid selected from the group consisting ofa, A, Aib, Bal, C, D, d, E, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T,V, W, and Y, such as an amino acid selected from the group consisting ofa, A, Aib, Bal, C, d, E, D, F, G, H, K, k, L, M, N, Nmg, p, Q, R, S, T,and Y (e.g., the amino acid is a, Aib, D, d, G, H, K, k, N, Nmg, p, Q,R, A, E, C, or M);

X1006 is optionally an amino acid selected from the group consisting ofA, Aib, Btq, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y,such as an amino acid selected from the group consisting of A, Btq, C,D, G, I, K, H, L, M, N, Q, R, S, V, and Y (e.g., an amino acid selectedfrom the group consisting of A, C, D, G, H, K, N, Q, R, S, and M);

X1008 is optionally an amino acid selected from the group consisting ofF, H, K, W, and Y, such as an amino acid selected from the groupconsisting of F, H, and Y;

X1010 is optionally an amino acid selected from the group consisting ofA, Aib, C, D, E, F, G, H, I, K, L, M, N, Nmf, P, Q, R, S, T, V, W, andY, such as an amino acid selected from the group consisting of A, D, E,F, G, H, K, L, M, N, P, Q, R, S, T, V, W, and Y (e.g., an amino acidselected from the group consisting of A, D, E, K, M, N, Q, R, F, H, P,S, V, W, and Y);

X1014 is optionally an amino acid selected from the group consisting ofA, Aib, Bhe, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, andY, such as an amino acid selected from the group consisting of A, Aib,Bhe, C, D, E, H, I, K, L, M, N, Q, R, S, T, V, W, and Y (e.g., A, C, D,E, K, L, M, N, Q, R, T, V, or Aib);

X1017 is optionally an amino acid selected from the group consisting of(omega-methyl)-R, A, Aib, Bhr, C, Cha, Cit, D, Dab, Dap, E, Eag, Eew, F,G, H, Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, Q, R, S, T, V,W, and Y, such as an amino acid selected from the group consisting of(omega-methyl)-R, A, Aib, Bhr, C, Cha, Cit, Dab, Dap, Eag, Eew, F, H,Har, Hci, Hle, I, K, L, M, N, Nle, Nva, Opa, Orn, R, S, T, V, and Y(e.g., an amino acid selected from the group consisting of A, Aib, C,Cha, Dab, Dap, Eag, Eew, H, Har, Hci, Hle, K, Nle, Nva, Opa, Orn, R, I,L, S, and M); and/or

X1018 is optionally an amino acid selected from the group consisting ofA, Bal, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y, suchas an amino acid selected from the group consisting of A, C, D, E, F, I,K, L, M, N, Q, R, V, and W (e.g., an amino acid selected from the groupconsisting of A, L, N, M, and R).

In some embodiments, the peptide of the invention comprises one or moreadditional amino acid residues attached to the N- or C-terminus of theamino acid sequence. For example, the peptide comprising the structureof any one of formulas (I)-(III), in some embodiments, further comprisesone or more N-terminal amino acid(s) directly linked to X1001, whereinthe N-terminal amino acid(s) comprise the amino acid sequence selectedfrom the group consisting of

X1000,

X999-X1000,

X998-X999-X1000,

X997-X998-X999-X1000 (SEQ ID NO: 3123),

X996-X997-X998-X999-X1000 (SEQ ID NO: 3124),

X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3125),

X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3126),

X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3127),

X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3128),

X991-X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO: 3129),and

X990-X991-X992-X993-X994-X995-X996-X997-X998-X999-X1000 (SEQ ID NO:3130).

When the peptide comprises one or more N-terminal amino acids, X1000 isA or K; X999 is V or K; X998 is Q or K; X997 is L or K; X996 is R or K;X995 is G or K; X994 is V or K; X993 is G or K; X992 is S or K; X991 isK; and X990 is K.

In addition to the core structures set forth in formulas (I)-(III),other structures that are specifically contemplated are those in whichone or more additional amino acids are attached to the C-terminus of thecore structure directly linked to X1020. For example, the C-terminaladdition optionally comprises an amino acid sequence selected from thegroup consisting of X1021, X1021-X1022, X1021-X1022-X1023, andX1021-X1022-X1023-X1024 (SEQ ID NO: 3131), wherein X1021 is T or K;X1022 is S or K; and X1023 and X1024 are K.

The invention further includes a TFPI-binding peptide comprising orconsisting of an amino acid sequence having at least 60% identity (e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identity) to the amino acid sequenceAc-FQSK-Nmg-NVFVDGYFERL-Aib-AKL-NH2 (formula IV) (SEQ ID NO: 164). Insome instances, the peptide comprises or consists of the amino acidsequence of any one of formulas (I)-(III), as described herein. Theinvention also includes a peptide comprising or consisting of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 8-978(e.g., a peptide comprising or consisting of the amino acid sequenceselected from the group consisting of SEQ ID NOs: 8-741 and 962-972(such as SEQ ID NOs: 8-741, 962-968, 971, or 972) and/or selected fromthe group consisting of 742-961 (such as SEQ ID NOs: 744-961) and/orselected from the group consisting of SEQ ID NOs: 973-978).

The invention includes peptides that comprise a cyclic structure. Inthis regard, the invention includes peptides comprising cyclicstructures within the peptide (e.g., one or more loops formed by linkagebetween amino acids other than the N- and C-terminal amino acids),peptides comprising a cyclic structure formed by the interaction of aterminal amino acid with an amino acid within the peptide sequence, andpeptides cyclized head to tail. The peptide may also be part of a largercyclic structure formed by surrounding additional amino acids orchemical substituents. The peptides of the invention, in some instances,comprise intramolecular disulfide bonds. In some embodiments, theintramolecular disulfide bonds are formed by cysteine residues. Peptidescomprising cyclic structures formed by non-cysteine residues, or anon-cysteine residue and a cysteine residue, also are provided. Forexample, in one embodiment, the inventive peptide comprises at least onenon-conventional amino acid or chemical moiety that mediatescyclization. Suitable non-conventional amino acids or chemical moietiesinclude, but are not limited to, FA19205, FA19204, FA19203, FA03202,Hcy, hcy, Cea, and c. The amino acids or moieties responsible forcyclization are sufficiently spaced apart to allow formation of a loopstructure, e.g., the amino acids or moieties are separated by two,three, four, five, six, seven, eight, or more residues.

In one aspect, the peptide comprising the structure of formulas(I)-(III) contains at least two cysteine residues (e.g., the peptidecontains two cysteine residues) that are spaced apart by at least threeamino acid residues such that the cysteines form an intramoleculardisulfide bond. In some instances, the cysteines are spaced apart bymore than three amino acid residues. For example, in the peptidecomprising the structure of formulas (I), (II), or (III), any two ofX1000, X1001, X1003, X1004, X1005, X1006, X1010, X1011, X1013, X1014,X1017, X1018, X1020 and X1021 are optionally cysteines capable offorming a disulfide bridge. Accordingly, in some aspects, the peptidecontains two cysteine residues: one of X1000, X1005, X1010 and X1014 iscysteine, and one of X1006, X1010, X1017 and X1021 is a cysteine. Theinvention contemplates all of the possible combinations of cysteinepairs, e.g., X1000 and X1006 are C; X1000 and X1010 are C; X1000 andX1017 are C; X1005 and X1017 are C; X1010 and X1017 are C; X1010 andX1021 are C; or X1014 and X1021 are C. Other exemplary cyclic peptidesof the invention include, e.g., JBT2441, JBT2450, JBT2466-JBT2469,JBT2489-JBT2495, JBT2497-JBT2499, and JBT2513-JBT2518 (SEQ ID NOs: 4159,4167, 4181-4184, 4204-4210, 4212-4214, and 4228-4233, respectively).

The invention further provides a peptide that binds TFPI, the peptidecomprising the structure of formula (V):X2001-X2002-X2003-X2004-X2005-X2006-[X2007-X2008-X2009-X2010-X2011-X2012-X2013-X2014-X2015-X2016-X2017-X2018]-X2019-X2020-X2021-X2022-X2023(V) (SEQ ID NO: 3118), wherein the peptide forms a cyclic structuregenerated by a linkage, e.g., a disulfide bond, between X2007 and X2018(denoted as brackets within formula (V)). In formula (V), X2001, X2002,and X2023 are independently either present or absent. When present,X2001 is an amino acid selected from the group consisting of A, D, E, F,G, H, I, K, L, P, R, S, T, V, and W; X2002 an amino acid selected fromthe group consisting of A, D, E, F, G, H, I, K, L, M, P, R, S, T, V, andW; and X2023 is an amino acid selected from the group consisting of A,D, E, F, G, I, K, L, R, S, T, V, W, and Y. In addition,

X2003 is an amino acid selected from the group consisting of A, F, I, K,L, R, S, T, V, W, and Y;

X2004 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, R, S, T, V, and W;

X2005 is W;

X2006 is an amino acid selected from the group consisting of F, H, I, K,L, R, V, and W;

X2007 is an amino acid selected from the group consisting of C, Hcy,Dap, and K (e.g., C or Hcy);

X2008 is an amino acid selected from the group consisting of A, G, R, S,and T;

X2009 is an amino acid selected from the group consisting of a, A, I, K,L, M, m, Nle, p, R, Sem, and V;

X2010 is an amino acid selected from the group consisting of A, G, I, K,L, P, R, S, T, and V;

X2011 is an amino acid selected from the group consisting of D, E, G, S,and T;

X2012 is an amino acid selected from the group consisting of A, a, D, d,E, e, F, f, G, I, K, k, L, l, M, m, Nle, nle, P, p, R, r, S, s, Sem, T,t, V, v, W, and w;

X2013 is an amino acid selected from the group consisting of A, D, d, E,e, F, G, I, K, L, R, S, s, T, V, and W;

X2014 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, M, R, S, T, V, and W;

X2015 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, M, Nle, R, S, T, V, and W;

X2016 is an amino acid selected from the group consisting of A, D, E, F,I, K, L, M, Nle, R, S, Sem, T, V, W, and Y;

X2017 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, R, S, T, V, W, and Y;

X2018 is an amino acid selected from the group consisting of C and D(e.g., X2018 is C);

X2019 is an amino acid selected from the group consisting of A, F, I, L,S, T, V, and W;

X2020 is an amino acid selected from the group consisting of F and W;

X2021 is an amino acid selected from the group consisting of I, L, andV; and

X2022 is an amino acid selected from the group consisting of A, D, E, F,G, I, K, L, P, R, S, T, V, and W.

In some instances, in the peptide of the invention comprising thestructure of formula (V),

X2001 is optionally an amino acid selected from the group consisting ofA, D, F, G, H, K, L, P, and S, such as an amino acid selected from thegroup consisting of A, D, F, G, H, K, L, and S (when X2001 is present);

X2002 is optionally an amino acid selected from the group consisting ofA, D, F, G, H, K, L, P, R, and S, such as an amino acid selected fromthe group consisting of A, F, H, K, L, M, R, and S (e.g., H, F, M or R)(when X2002 is present);

X2003 is optionally an amino acid selected from the group consisting ofA, F, K, L, S, and Y, such as an amino acid selected from the groupconsisting of F, S, and Y (e.g., F or Y);

X2004 is optionally an amino acid selected from the group consisting ofA, D, F, G, K, L, and S (e.g., K);

X2005 is optionally W;

X2006 is optionally an amino acid selected from the group consisting ofF, H, K, and L (e.g., F or H);

X2007 is optionally an amino acid selected from the group consisting ofC and HcY (e.g., X2007 is C);

X2008 is optionally an amino acid selected from the group consisting ofA, G, and S;

X2009 is optionally an amino acid selected from the group consisting ofa, A, K, L, V, M, m, Nle, Sem, and p, such as an amino acid selectedfrom the group consisting of M, Nle, p, and V (e.g., M, Sem, or V);

X2010 is optionally an amino acid selected from the group consisting ofA, G, K, L, P, R, and S, such as an amino acid selected from the groupconsisting of A, K, L, P, R and S (e.g., K, P, or R);

X2011 is optionally an amino acid selected from the group consisting ofD, G, and S (e.g., D or S);

X2012 is optionally an amino acid selected from the group consisting ofA, a, D, d, F, f, G, K, k, L, l, M, m, Nle, P, S, and s, such as anamino acid selected from the group consisting of D, d, F, f, G, K, k, L,l, M, Nle, P, S, and Sem (e.g., an amino acid selected from the groupconsisting of F, L, l, Sem, and M);

X2013 is optionally an amino acid selected from the group consisting ofA, D, d, F, G, K, L, S, and s, such as an amino acid selected from thegroup consisting of A, D, F, G, K, L and S (e.g., D, G, K, or S);

X2014 is optionally an amino acid selected from the group consisting ofD, F, G, K, L, and S (e.g., D or G);

X2015 is optionally an amino acid selected from the group consisting ofA, D, F, G, I, K, L, M, Nle, S, and T (e.g., I or T);

X2016 is optionally an amino acid selected from the group consisting ofD, F, K, L, M, Nle, S, and Y, such as an amino acid selected from thegroup consisting of D, F, K, L, M, Nle, S, Sem, and Y (e.g., D, F, M,Sem, or Y);

X2017 is optionally an amino acid selected from the group consisting ofA, D, F, G, K, L, S, T, and Y (e.g., S or T);

X2018 is optionally C;

X2019 is optionally an amino acid selected from the group consisting ofA, F, L, S, and V (e.g., A or V);

X2020 is optionally an amino acid selected from the group consisting ofF and W (e.g., W);

X2021 is optionally an amino acid selected from the group consisting ofL and V (e.g., V);

X2022 is optionally an amino acid selected from the group consisting ofA, D, F, G, K, L, P, R, S, and W, such as an amino acid selected fromthe group consisting of A, F, G, K, L, P, R, S, and W (e.g., an aminoacid selected from the group consisting of F, L, K, R, P, and W); and

X2023 is optionally an amino acid selected from the group consisting ofA, D, F, G, K, L, M, S, and Y, such as an amino acid selected from thegroup consisting of A, D, F, G, L M, S, and Y (e.g., an amino acidsequence selected from the group consisting of A, D, F, M, S and Y)(when X2023 is present).

The invention further includes a peptide that binds TFPI, wherein thepeptide comprises the structure of formula (VI):X2001-X2002-F/Y-K-W-F/H-[C-X2008-M/V-X2010-D-X2012-X2013-G-I/T-X2016-SIT-C]-A/V-W-V-X2022-X2023(VI) (SEQ ID NO: 3119). In the peptide comprising the structure offormula (VI), X2001, X2002 and X2023 are each independently present orabsent. If X2001, X2002, and/or X2023 are present, any of X2001, X2002and X2023 is independently selected from any amino acid. In addition,X2008, X2010, X2012, X2013, X2016, and X2022 are each independentlyselected from any amino acid.

In some aspects, in the peptide of formula (VI),

X2001 is optionally an amino acid selected from the group consisting ofA, D, E, F, G, H, I, K, L, P, R, S, T, V, and W, such as an amino acidselected from the group consisting of A, D, F, G, H, K, L, P, and S(e.g., an amino acid selected from the group consisting of A, D, F, G,H, K, L, and S) (when X2001 is present);

X2002 is optionally an amino acid selected from the group consisting ofA, D, E, F, G, H, I, K, L, M, P, R, S, T, V, and W, such as an aminoacid selected from the group consisting of A, D, F, G, H, K, L, M, P, R,and S (e.g., an amino acid selected from the group consisting of A, F,H, K, L, M, R, and S, such as H, F, M, or R) (when X2002 is present);

X2008 is optionally an amino acid selected from the group consisting ofA, G, R, S, and T, such as an amino acid selected from the groupconsisting of A, G, and S;

X2010 is optionally an amino acid selected from the group consisting ofA, G, I, K, L, P, R, S, T, and V, such as an amino acid selected fromthe group consisting of A, G, K, L, P, R, and S (e.g., an amino acidselected from the group consisting of A, K, L, P, R, and S, such as K, Por R);

X2012 is optionally an amino acid selected from the group consisting ofA, a, D, d, E, e, F, f, G, I, I, K, k, L, l, M, m, Nle, nle, P, p, R, r,S, s, Sem, T, t, V, v, W, and w, such as an amino acid selected from thegroup consisting of A, a, D, d, F, f, G, K, k, L, l, M, m, Nle, P, S, s,and Sem (e.g., an amino acid selected from the group consisting of D, d,F, f, G, K, k, L, l, M, Nle, P, S, and Sem, such as F, L, l, Sem, or M);

X2013 is optionally an amino acid selected from the group consisting ofA, D, d, E, e, F, G, I, K, L, R, S, s, T, V, and W, such as an aminoacid selected from the group consisting of A, D, d, F, G, K, L, S, and s(e.g., an amino acid selected from the group consisting of A, D, F, G,K, L, and S, such as D, G, K, or S);

X2016 is optionally an amino acid selected from the group consisting ofA, D, E, F, I, K, L, M, Nle, R, S, Sem, T, V, W, and Y, such as an aminoacid selected from the group consisting of D, F, K, L, M, Nle, S, Sem,and Y (e.g., an amino acid selected from the group consisting of D, F,K, L, M, Nle, S, and Sem, such as F, Sem, or M);

X2022 is optionally an amino acid selected from the group consisting ofA, D, E, F, G, I, K, L, P, R, S, T, V, and W, such as an amino acidselected from the group consisting of A, D, F, G, K, L, P, R, S, and W(e.g., an amino acid selected from the group consisting of A, F, G, K,L, P, R, S, and W, such as F, L, K, R, P, or W); and/or

X2023 is optionally an amino acid selected from the group consisting ofA, D, E, F, G, I, K, L, R, M, S, T, V, W, and Y, such as an amino acidselected from the group consisting of A, D, F, G, K, L, M, S, and Y(e.g., an amino acid selected from the group consisting of A, D, F, G, LM, S, and Y, such as A, D, F, M, S, or Y) (when X2023 is present).

The TFPI-binding peptide of the invention, in one aspect, comprises anamino acid sequence having at least 60% identity (e.g., at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identity) to the sequence of formulaVII: Ac-FYYKWH[CGMRDMKGTMSC]AWVKF-NH2 (VII) (SEQ ID NO: 1040).Optionally, the peptide comprises or consists of the amino acid sequenceof formula (V)-(VII) as defined herein. The invention also includes apeptide comprising or consisting of the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1001-1293 (e.g., a peptidecomprising or consisting of the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1001-1212 and 1290-1291 (such as SEQ IDNOs: 1001-120, 1290, or 1291) and/or selected from the group consistingof SEQ ID NOs: 1213-1289 and/or selected from the group consisting of1292 and 1293).

The invention further provides a TFPI-binding peptide comprising atleast amino acids 3-21 (X3003-X3021) of the structure of formula (VIII):X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-X3012-X3013-X3014-X3015-X3016-X3017-X3018-X3019-X3020-X3021(VIII) (SEQ ID NO: 3120). In formula (VIII), X3001 and X3002 areindependently either present or absent in the peptide. If present, X3001is an amino acid selected from the group consisting of A, C, D, F, G, I,K, L, M, N, P, Q, R, S, T, W, E, H, and Y; and X3002 is an amino acidselected from the group consisting of A, C, D, F, H, K, M, N, P, R, S,T, W, Y, G, I, and L. In addition,

X3003 is an amino acid selected from the group consisting of A, C, D, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y;

X3004 is an amino acid selected from the group consisting of A, C, D, E,F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P;

X3005 is an amino acid selected from the group consisting of C, D, F, G,H, I, K, L, M, N, P, R, S, T, V, W, and Y;

X3006 is an amino acid selected from the group consisting of A, W, C, K,P, R, and H;

X3007 is an amino acid selected from the group consisting of Q, A, C, F,G, H, I, K, L, N, R, S, T, W, and Y;

X3008 is an amino acid selected from the group consisting of A, C, F, G,H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I;

X3009 is an amino acid selected from the group consisting of A, C, F, G,H, I, L, M, R, S, T, V, W, Y, and K;

X3010 is an amino acid selected from the group consisting of A, C, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

X3011 is an amino acid selected from the group consisting of A, G, I, K,L, M, N, Q, R, S, T, V, W, Y, C, F, and H;

X3012 is an amino acid selected from the group consisting of A, C, H, I,K, L, and R;

X3013 is an amino acid selected from the group consisting of A, C, F, G,H, K, L, M, R, S, V, W, Y, and I;

X3014 is an amino acid selected from the group consisting of A, C, F, G,H, I, L, M, N, Q, R, S, T, V, W, Y, and K;

X3015 is an amino acid selected from the group consisting of A, K, andR;

X3016 is an amino acid selected from the group consisting of A, F, K,and R;

X3017 is an amino acid selected from the group consisting of A, C, F, G,I, K, L, N, Q, R, S, T, V, W, Y, H, A, and M;

X3018 is an amino acid selected from the group consisting of A, C, F, I,K, L, M, Q, R, V, W, and Y;

X3019 is an amino acid selected from the group consisting of A, C, D, E,F, G, H, K, L, N, P, Q, R, V, W, Y, and I;

X3020 is an amino acid selected from the group consisting of A, C, F, G,H, K, L, M, N, Q, R, V, W, Y, I, and P; and

X3021 is an amino acid selected from the group consisting of A, C, H, I,K, L, M, N, P, Q, R, T, V, W, Y, F, and G.

In some aspects of the invention, the peptide comprises the sequence offormula (VIII), wherein

X3001 is optionally an amino acid selected from the group consisting ofA, C, D, G, I, K, L, M, N, P, Q, R, S, T, W, E, H, and Y, such as anamino acid selected from the group consisting of A, C, D, G, K, L, M, N,P, R, S, T, E, H, and Y (when X3001 is present);

X3002 is optionally an amino selected from the group consisting of C, F,H, K, R, S, W, Y, G, I, and L, such as an amino acid selected from thegroup consisting of C, K, R, W, Y, G, I, and L (when X3002 is present);

X3003 is optionally an amino acid selected from the group consisting ofA, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, and W, such as an aminoacid selected from the group consisting of A, C, G, H, I, K, L, M, R, S,T, and W;

X3004 is optionally an amino acid selected from the group consisting ofA, C, D, G, H, I, K, L, M, N, R, S, T, V, and P, such as an amino acidselected from the group consisting of A, C, G, H, I, K, L, M, N, R, S,T, and P;

X3005 is optionally an amino acid selected from the group consisting ofC, F, H, I, K, M, R, T, W, and Y, such as an amino acid selected fromthe group consisting of C, F, H, K, R, and W;

X3006 is optionally an amino acid selected from the group consisting ofP, H, and A;

X3007 is optionally an amino acid selected from the group consisting ofC, G, R, W, A, and L, such as an amino acid selected from the groupconsisting of L, C, R, and W;

X3008 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, K, L, M, N, Q, R, T, V, W, Y, and I, such as an aminoacid selected from the group consisting of A, C, F, H, K, R, V, W, Y,and I;

X3009 is optionally an amino acid selected from the group consisting ofC, I, R, V, and K, such as an amino acid selected from the groupconsisting of C, R, V, and K;

X3010 is optionally an amino acid selected from the group consisting ofA, C, G, H, I, K, L, M, Q, R, S, and T, such as an amino acid selectedfrom the group consisting of A, C, K, L, Q, R, and S;

X3011 is optionally an amino acid selected from the group consisting ofA, I, K, L, M, R, S, V, W, C, F, and H, such as an amino acid selectedfrom the group consisting of I, K, L, M, R, V, W, C, F, and H;

X3012 is optionally an amino acid selected from the group consisting ofH and R (e.g., H);

X3013 is optionally an amino acid selected from the group consisting ofC, F, K, L, M, R, V, and I, such as an amino acid selected from thegroup consisting of C, K, R, V, and I;

X3014 is optionally an amino acid selected from the group consisting ofA, M, C, F, H, I, L, N, R, S, V, W, and K, such as an amino acidselected from the group consisting of A, S, C, F, H, I, R, and K;

X3015 is optionally K or R;

X3016 is optionally K or R;

X3017 is optionally an amino acid selected from the group consisting ofA, C, F, G, I, K, L, N, Q, R, S, T, V, W, H, A, and M, such as an aminoacid selected from the group consisting of C, G, I, K, L, N, Q, R, S, T,V, H, A, and M;

X3018 is optionally an amino acid selected from the group consisting ofA, K, C, I, L, R, and W (e.g., K, C, I, R, or W);

X3019 is optionally an amino acid selected from the group consisting ofA, C, E, H, K, N, Q, R, and I, such as an amino acid selected from thegroup consisting of C, E, H, K, R, and I;

X3020 is optionally an amino acid selected from the group consisting ofC, H, L, M, R, V, I, and P (e.g., C, M, I, or P); and

X3021 is optionally an amino acid selected from the group consisting ofA, C, H, I, K, L, M, N, Q, R, V, W, Y, F, and G, such as an amino acidselected from the group consisting of A, C, H, I, K, L, M, N, Q, R, V,W, F, and G.

The invention further provides a peptide that binds TFPI and comprisesat least amino acids 3-21 (X3003-X3021) of the structure of formula(IX):X3001-X3002-X3003-X3004-X3005-X3006-X3007-X3008-X3009-X3010-X3011-H-X3013-X3014-K/R-R-X3017-X3018-X3019-X3020-X3021(IX) (SEQ ID NO: 3121). In formula (IX), X3001 and X3002 areindependently either present or absent in the peptide. If present, X3001and/or X3002 are independently selected from any amino acid. Likewise,X3003, X3004, X3005, X3006, X3007, X3008, X3009, X3010, X3011, X3013,X3014, X3017, X3018, X3019, X3020 and X3021 are each independentlyselected from any amino acid. When present, X3001 is optionally an aminoacid selected from the group consisting of A, C, D, F, G, I, K, L, M, N,P, Q, R, S, T, W, E, H, and Y, such as an amino acid selected from thegroup consisting of A, C, D, G, I, K, L, M, N, P, Q, R, S, T, W, E, H,and Y (e.g., an amino acid selected from the group consisting of A, C,D, G, K, L, M, N, P, R, S, T, E, H, and Y). Likewise, when present,X3002 is optionally an amino acid selected from the group consisting ofA, C, D, F, H, K, M, N, P, R, S, T, W, Y, G, I, and L, such as an aminoacid selected from the group consisting of C, F, H, K, R, S, W, Y, G, I,and L (e.g., an amino acid selected from the group consisting of C, K,R, W, Y, G, I, and L). Also with respect to formula (IX),

X3003 is optionally an amino acid selected from the group consisting ofA, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, and Y, such as anamino acid selected from the group consisting of A, C, D, F, G, H, I, K,L, M, N, P, Q, R, S, T, and W (e.g., an amino acid selected from thegroup consisting of A, C, G, H, I, K, L, M, R, S, T, and W);

X3004 is optionally an amino acid selected from the group consisting ofA, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and P, such asan amino acid selected from the group consisting of A, C, D, G, H, I, K,L, M, N, R, S, T, V, and P (e.g., an amino acid selected from the groupconsisting of A, C, G, H, I, K, L, M, N, R, S, T, and P);

X3005 is optionally an amino acid selected from the group consisting ofC, D, F, G, H, I, K, L, M, N, P, R, S, T, V, W, and Y, such as an aminoacid selected from the group consisting of C, F, H, I, K, M, R, T, W,and Y (e.g., an amino acid selected from the group consisting of C, F,H, K, R, and W);

X3006 is optionally an amino acid selected from the group consisting ofA, W, C, K, P, R and H, such as an amino acid selected from the groupconsisting of P, H, and A;

X3007 is optionally an amino acid selected from the group consisting ofQ, A, C, F, G, H, I, K, L, N, R, S, T, W, and Y, such as an amino acidselected from the group consisting of C, G, R, W, A, and L (e.g., L, C,R, or W);

X3008 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y, and I, such as anamino acid selected from the group consisting of A, C, F, G, H, K, L, M,N, Q, R, T, V, W, Y, and I (e.g., an amino acid selected from the groupconsisting of A, C, F, H, K, R, V, W, Y, and I);

X3009 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, I, L, M, R, S, T, V, W, Y, and K, such as an amino acidselected from the group consisting of C, I, R, V, and K (e.g., C, R, V,or K);

X3010 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, such as anamino acid selected from the group consisting of A, C, G, H, I, K, L, M,Q, R, S, and T (e.g., an amino acid selected from the group consistingof A, C, K, L, Q, R, and S);

X3011 is optionally an amino acid selected from the group consisting ofA, G, I, K, L, M, N, Q, R, S, T, V, W, Y, C, F, and H, such as an aminoacid selected from the group consisting of A, I, K, L, M, R, S, V, W, C,F, and H (e.g., an amino acid selected from the group consisting of I,K, L, M, R, V, W, C, F, and H);

X3013 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, K, L, M, R, S, V, W, Y, and I, such as an amino acidselected from the group consisting of C, F, K, L, M, R, V, and I (e.g.,C, K, R, V, or I);

X3014 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, and K, such as an aminoacid selected from the group consisting of A, M, C, F, H, I, L, N, R, S,V, W, and K (e.g., an amino acid selected from the group consisting ofA, S, C, F, H, I, R, and K);

X3017 is optionally an amino acid selected from the group consisting ofA, C, F, G, I, K, L, N, Q, R, S, T, V, W, Y, H, A, and M, such as anamino acid selected from the group consisting of A, C, F, G, I, K, L, N,Q, R, S, T, V, W, H, A, and M (e.g., an amino acid selected from thegroup consisting of C, G, I, K, L, N, Q, R, S, T, V, H, A, and M);

X3018 is optionally an amino acid selected from the group consisting ofA, C, F, I, K, L, M, Q, R, V, W, and Y, such as an amino acid selectedfrom the group consisting of A, K, C, I, L, R, and W (e.g., K, C, I, R,or W);

X3019 is optionally an amino acid selected from the group consisting ofA, C, D, E, F, G, H, K, L, N, P, Q, R, V, W, Y, and I, such as an aminoacid selected from the group consisting of A, C, E, H, K, N, Q, R, and I(e.g., C, E, H, K, R, or I);

X3020 is optionally an amino acid selected from the group consisting ofA, C, F, G, H, K, L, M, N, Q, R, V, W, Y, I, and P, such as an aminoacid selected from the group consisting of C, H, L, M, R, V, I, and P(e.g., C, M, I, or P); and/or

X3021 is optionally an amino acid selected from the group consisting ofA, C, H, I, K, L, M, N, P, Q, R, T, V, W, Y, F, and G, such as an aminoacid selected from the group consisting of A, C, H, I, K, L, M, N, Q, R,V, W, Y, F, and G (e.g., an amino acid selected from the groupconsisting of A, C, H, I, K, L, M, N, Q, R, V, W, F, and G).

The TFPI-binding peptide of the invention comprises, in some aspects, anamino acid sequence having at least 60% identity (e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or 100% identity) to the sequence of formula (X):Ac-GYASFPWFVQLHVHKRSWEMA-NH2 (X) (SEQ ID NO: 223). Optionally, thepeptide comprises or consists of the amino acid sequence of formula(VIII)-(IX) as defined herein. As used herein, “at least 60% identity”and similar terms encompass any integer from, e.g., 60%, to 100%, suchas 60%, 61%, 62%, and the like. Also, the term “at least [percentage]identity” encompasses any percentage that is greater than or equal tothe number of identical amino acids divided by the total number of aminoacids of the peptide of the invention ([at least percentageidentity]≧[number of identical amino acids]/[total number of amino acidsof the peptide of the invention]).

The invention also includes a peptide comprising or consisting of theamino acid sequence selected from the group consisting of SEQ ID NOs:2001-2498 (e.g., a peptide comprising or consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 2001-2296 and2498 (such as SEQ ID NOs: 2001-2126, 2128-2296, or 2498) and/or selectedfrom the group consisting of SEQ ID NOs: 2297-2497 (such as SEQ ID NOs:2298-2497)). The invention further provides a peptide comprising orconsisting of the amino acid sequence selected from the group consistingof SEQ ID NOs: 3001-3108 (e.g., a peptide comprising or consisting ofthe amino acid sequence selected from the group consisting of SEQ IDNOs: 3001-3064 (such as SEQ ID NOs: 3001-3048, 3051-3053, 3055, or3057-3064) and/or selected from the group consisting of SEQ ID NOs:3065-3084 (such as SEQ ID NOs: 3066-3084) and/or selected from the groupconsisting of SEQ ID NOs: 3085-3108).

The peptide of SEQ ID NOs: 1-7 also, in some aspects, comprises one ormore amino acids attached at the N- or C-terminus of SEQ ID NOs: 1-7.For example, the invention includes a peptide comprising or consistingof the amino acid sequence of JBT0047, JBT0051, JBT0055, JBT0131,JBT0132, JBT0133, JBT0155, JBT0158, JBT0162, JBT0163, JBT0164, JBT0166,JBT0169, JBT0170, JBT0171, JBT0174, JBT0175, or JBT0293, all of whichcomprise the amino acid sequence of SEQ ID NO: 1. Exemplary peptidescomprising the amino acid sequence of SEQ ID NO: 2 include peptidescomprising or consisting of the amino acid sequence of JBT0294, JBT0295,JBT0296, JBT0297, JBT0298, JBT0299, JBT0300, JBT0301, JBT0302, JBT0303,JBT0304, JBT0305, JBT0306, JBT0307, JBT0308, JBT0309, JBT0310, orJBT0311. Exemplary peptides comprising the amino acid sequence of SEQ IDNO: 3 comprise or consist of the amino acid sequence of JBT0049,JBT0053, JBT0057, JBT0190, JBT0193, or JBT0197. The invention furtherincludes a peptide comprising or consisting of the amino acid sequenceof JBT0050, JBT0054, JBT0058, JBT0129, JBT0130, JBT0205, JBT0208,JBT0211, JBT0212, JBT0217, JBT0218, or JBT0219, all of which include theamino acid sequence of SEQ ID NO: 4. Exemplary peptides comprising SEQID NO: 5 include those comprising or consisting of the amino acidsequence of JBT0101, JBT0052, JBT0103, JBT0178, or JBT0182. Theinvention additionally includes a peptide comprising or consisting ofthe amino acid sequence of JBT0120, JBT0124, JBT0247, JBT0248, JBT0251,or JBT0252, each of which include the amino acid sequence of SEQ ID NO:6. A peptide including the amino acid sequence of SEQ ID NO: 7, e.g., apeptide comprising or consisting of the amino acid sequence of JBT0122,JBT0126. JBT0221, JBT0224, JBT0225, JBT0226, JBT0228, JBT0232, orJBT0233, also provided by the invention. The peptides described hereinare set forth in Table 5 of Example 1 and in FIGS. 12A-12P, 13A-13D,14A-14E, 15A-15D, 16A-16F, 17A-17B, and 18A-18E.

The invention further includes a TFPI-binding peptide comprising thestructure of formula (XI):X4001-Q-X4003-X4004-X4005-X4006-X4007-X4008-X4009-X4010-X4011-X4012-X4013-X4014-R-X4016-X4017-X4018-X4019-X4020(XI). With respect to formula (XI),

X4001 is an amino acid selected from the group consisting of F, L, M, Y,1Ni, Thi, Bta, and Dopa (e.g., F, Y, 1Ni, Bta, or Dopa);

X4003 is an amino acid selected from the group consisting of C, D, E, M,Q, R, S, T, Ede(O), and Cmc (e.g., D, E, or S);

X4004 is an amino acid selected from the group consisting of Aib, E, G,I, K, L, M, P, R, W, and Y (e.g., K);

X4005 is an amino acid selected from the group consisting of a, A, Aib,C, D, d, E, G, H, K, k, M, N, Nmg, p, Q, R, NpropylG, aze, pip, tic,oic, hyp, nma, Ncg, Abg, Apg, thz, and dtc (e.g., p, Nmg, NpropylG, aze,pip, tic, oic, or hyp);

X4006 is an amino acid selected from the group consisting of A, C,C(NEM), D, E, G, H, K, M, N, Q, R, S, V, Cit, C(Acm), Nle, I, Ede(O),Cmc, Ed, Eea, Eec, Eef, Nif, and Eew (e.g., C, E, K, R, S, V, C(Acm),Nle, C(NEM), I, or Cit);

X4007 is an amino acid selected from the group consisting of I, V, T,Chg, Phg, and Tle (e.g., V or Tle);

X4008 is an amino acid selected from the group consisting of F, H, 1Ni,2Ni, Pmy, and Y (e.g., H, 1Ni, 2Ni, or Pmy);

X4009 is an amino acid selected from the group consisting of Aib, V,Chg, Phg, Abu, Cpg, Tle, and L-2-amino-4,4,4-trifluorobutyric acid(e.g., V, Abu, or Tle);

X4010 is an amino acid selected from the group consisting of A, C, D, d,E, F, H, K, M, N, P, Q, R, S, T, V, W, Y, Nmd, and C(NEM) (e.g., D, P, Cor T);

X4011 is an amino acid selected from the group consisting of A, a, G, p,Sar, c, and hcy (e.g., G, a, c, hcy, or Sar);

X4012 is an amino acid selected from the group consisting of Y, Tym,Pty, Dopa, and Pmy (e.g., Y);

X4013 is an amino acid selected from the group consisting of C, F, 1Ni,Thi, and Bta (e.g., F, 1Ni, or Bta);

X4014 is an amino acid selected from the group consisting of A, Aib, C,C(NEM), D, E, K, L, M, N, Q, R, T, V, and Hcy (e.g., Aib, C, E, or Hcy);

X4016 is an amino acid selected from the group consisting of L, Hcy,Hle, and Aml;

X4017 is an amino acid selected from the group consisting of A, a, Aib,C, c, Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nva, Opa,Orn, R, S, Deg, Ebc, Eca, Egz, Aic, Apc, and Egt (e.g., A, Aib, C, c,Aic, Eca, or Deg);

X4018 is an amino acid selected from the group consisting of A, Aib,Hcy, hcy, C, c, L, Nle, M, N, and R (e.g., A, Aib, C, c, L, or Hcy);

X4019 is an amino acid selected from the group consisting of K, R, andHar (e.g., K); and

X4020 is an amino acid selected from the group consisting of K, L, Hcy,and Aml (e.g., L, Aml, and Hcy).

The TFPI-binding peptide of formula (XI) does not comprise the structureof formula (XII):X5001-Q-X5003-X5004-X5005-X5006-I/V-X5008-Aib/V-X5010-G-Y-X5013-X5014-R-L-X5017-X5018-K-K/L(XII). In formula (XII),

X5001 is an amino acid selected from the group consisting of F, L, M,and Y;

X5003 is an amino acid selected from the group consisting of C, D, E, M,Q, R, S, and T;

X5004 is an amino acid selected from the group consisting of E, G, I, K,L, M, P, R, W, and Y;

X5005 is an amino acid selected from the group consisting of a, A, Aib,C, D, d, E, G, H, K, k, M, N, Nmg, Q, R, and p;

X5006 is an amino acid selected from the group consisting of A, C, D, E,G, H, K, M, N, Q, R, S, and V;

X5008 is an amino acid selected from the group consisting of F, H, andY;

X5010 is an amino acid selected from the group consisting of A, C, D, E,F, H, D, M, N, P, Q, R, S, T, V, W, and Y;

X5013 is an amino acid selected from the group consisting of Aib, C, andF;

X5014 is an amino acid selected from the group consisting of A, Aib, C,D, E, K, L, M, N, Q, R, T, and V;

X5017 is an amino acid selected from the group consisting of A, Aib, C,Cha, Dab, Eag, Eew, H, Har, Hci, Hle, I, K, L, M, Nle, Nve, Opa, Orn, R,and S; and

X5018 is an amino acid selected from the group consisting of A, C, L, M,N, and R.

In one aspect, the TFPI-binding peptide of formula (XI) furthercomprises N-terminal amino acid(s) and/or moieties linked to X4001. TheN-terminal amino acid(s) and/or moieties are optionally selected fromthe group consisting of FAM-Ttds, a proline-glutamate tag (“PE”), Palm,2-phenyl acetyl, 3-phenyl propionyl, 2-(naphtha-2-yl)acetyl, hexanoyl,2-methyl propionyl, 3-methyl butanoyl, 2-naphthylsulfonyl, and1-naphthylsulfonyl. Alternatively or in addition, the TFPI-bindingpeptide of formula (XI) further comprises one or more amino acid(s)and/or moieties linked to X4020. The C-terminal amino acid(s) and/ormoieties are designated herein as X4021 and are optionally selected fromthe group consisting of C, c, C(NEM), K(Ttds-maleimidopropionyl(EtSH)),FA19205, FA19204, FA19203, FA03202, K(Tdts-maleimid), K(AOA), and Cea.

In one embodiment, the peptide comprises a cyclic structure formedbetween X4018 and X4021. In this regard, X4018 is optionally C or c, andX4021 is optionally Cea. In another embodiment, the peptide comprises acyclic structure formed between X4011 and X4014. In this regard, X4011is optionally c or hcy, and X4014 is optionally C or Hcy.

The invention also includes a peptide consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 4022, 4024,4032, 4036-4047, 4049-4078, 4086-4097, 4100-4127, 4129-4170, 4173-4195,4200-4214, 4217-4225, 4228, 4230, 4231, 4238, and 4239, as well as apeptide consisting of the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1294-1336, 4002, 4013, 4021, 4023, 4025-4031,4033-4035, 4048, 4079-4085, 4098, 4099, 4128, 4171, 4172, 4196-4199,4215, 4216, 4226, 4277, 4229, 4232, and 4233.

In certain embodiments, the peptide of the invention comprises orconsists of the amino acid sequence of JBT0047, JBT0049, JBT0101,JBT0120, or JBT0122 or any of the inventive peptides described herein(e.g., a peptide comprising or consisting of the amino acid sequence ofany one of SEQ ID NOs: 1-3108, such as a peptide comprising orconsisting of the amino acid sequence of any one of SEQ ID NOs: 8-741,744-968, 971-978, 1001-1210, 1213-1289, 1290-1293, 2001-2126, 2128-2296,2298-2498, 3001-3048, 3051-3053, 3055, 3057-3064, and 3067-3108; apeptide comprising or consisting of the amino acid sequence of any oneof SEQ ID NOs: 4022, 4024, 4032, 4036-4047, 4049-4078, 4086-4097,4100-4127, 4129-4170, 4173-4195, 4200-4214, 4217-4225, 4228, 4230, 4231,4238, and 4239; or a peptide comprising or consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 1294-1336,4002, 4013, 4021, 4023, 4025-4031, 4033-4035, 4048, 4079-4085, 4098,4099, 4128, 4171, 4172, 4196-4199, 4215, 4216, 4226, 4277, 4229, 4232,and 4233), or a variant of any of the foregoing. By “variant” is meant apeptide comprising one or more amino acid substitutions, amino aciddeletions, or amino acid additions to a parent amino acid sequence.Variants include, but are not limited to, peptides having an amino acidsequence that is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of theamino acid sequences provided herein while retaining the ability to bindTFPI and/or inhibit TFPI activity. In one embodiment, the peptidecomprises or consists of the amino acid sequence of JBT0132, JBT0303,JBT0193, JBT0178, JBT0120, or JBT0224.

In one aspect, the peptide of the invention consists of 40 amino acidsor less, such as 35 amino acids or less. Optionally, the peptide of theinvention consists of 25 amino acids or less, or 10 amino acids or less.In various embodiments, the peptide comprises 15-35 amino acid residues(e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, or 35 amino acid residues). However, it is alsocontemplated that a peptide described herein comprising one or moredeletions is suitable in the context of the invention so long as thepeptide binds TFPI and, optionally, blocks TFPI inhibition of thecoagulation cascade. In some aspects, amino acids are removed fromwithin the amino acid sequence, at the N-terminus, and/or at theC-terminus. Such peptide fragments can comprise 3-14 amino acid residues(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues).

Optionally, the peptide of the invention comprises one or more aminoacid substitutions (with reference to any of the amino acid sequencesprovided herein) that do not destroy the ability of the peptide to bindand/or inhibit TFPI. For instance, peptides comprising or consisting ofthe amino acid sequence selected from the group consisting of JBT0294,JBT0295, JBT0296, JBT0297, JBT0298, JBT0299, JBT0300, JBT0301, JBT0302,JBT0303, JBT0304, JBT0305, JBT0306, JBT0307, JBT0308, JBT0309, JBT0310,or JBT0311 are substitutional mutants of the amino acid sequence ofJBT0293 (the amino acid sequence of SEQ ID NO: 1 directly linked to aphenylalanine residue at the N-terminus and a lysine reside at theC-terminus) (see FIG. 4).

Amino acid substitutions include, but are not limited to, those which:(1) reduce susceptibility to proteolysis, (2) reduce susceptibility tooxidation, (3) alter binding affinities, and/or (4) confer or modifyother physiochemical or functional properties on a peptide. In oneaspect, the substitution is a conservative substitution, wherein anamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined within the art, and include amino acids withbasic side chains (e.g., lysine, arginine, and histidine), acidic sidechains (e.g., aspartic acid and glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan), beta-branched side chains (e.g., threonine, valine, andisoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, and histidine). It will be appreciated, however, that apractitioner is not limited to creating conservative substitutions solong as the resulting peptide retains the ability to downregulate, inwhole or in part, TFPI activity. The invention also embracesTFPI-inhibitory peptides comprising atypical, non-naturally occurringamino acids, which are well known in the art. Exemplary non-naturallyoccurring amino acids include ornithine, citrulline, hydroxyproline,homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyricacid, α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyricacid, y-amino butyric acid, 2-amino isobutyric acid, 3-amino propionicacid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, a fluoro-amino acid, a 3-methyl amino acid, α-C-methyl aminoacid, a N-methyl amino acid, 2-amino-isobutyric acid, β-homoglutamaticacid, β-homophenylalanine, β-homolysine, β-homoleucine,β-homoasparagine, β-homoglutamine, β-homoarginine, β-homoserine,β-homotyrosine, β-homoaspartic acid, β-homovaline, β-homoasparagin,(S)-cyclohexylalanine, (S)-citrullin, (S)-2,4-diaminobutyric acid,(S)-2,4-diaminobutyric acid, (S)-diaminopropionic acid,(S)-2-propargylglycine, (S)—N(omega)-nitro-arginine,L-homophenylalanine, S)-homo-arginine, (S)-homo-citrulline,(S)-homo-cysteine, (S)-2-amino-5-methyl-hexanoic acid, (S)-homo-lysine,(S)-norleucine, (S)—N-methylalanine, (S)—N-methyl-aspartic acid,(S)—N-methyl-glutamic acid, (S)—N-methyl-phenylalanine,N-methyl-glycine, (S)—N-methyl-lysine, (S)—N-methyl-leucine,(S)—N-methyl-arginine, (S)—N-methyl-serine, (S)—N-methyl-valine,(S)—N-methyl-tyrosine, (S)-2-amino-pentanoic acid,(S)-2-pyridyl-alanine, (S)-ornithine, L-phenylglycin, 4-phenyl-butyricacid and selenomethionine. The individual amino acids may have either Lor D stereochemistry when appropriate, although the L stereochemistry istypically employed for all of the amino acids in the peptide.

The invention further includes TFPI-inhibitory peptide variantscomprising one or more amino acids inserted within an amino acidsequence provided herein and/or attached to the N-terminus orC-terminus. In one aspect, the peptide further comprises one or moreamino acids that facilitate synthesis, handling, or use of the peptide,including, but not limited to, one or two lysines at the N-terminusand/or C-terminus to increase solubility of the peptide. Suitable fusionproteins include, but are not limited to, proteins comprising aTFPI-inhibitory peptide linked to one or more polypeptides, polypeptidefragments, or amino acids not generally recognized to be part of theprotein sequence. In one aspect, a fusion peptide comprises the entireamino acid sequences of two or more peptides or, alternatively,comprises portions (fragments) of two or more peptides. In addition toall or part of the TFPI-inhibitory peptides described herein, a fusionprotein optionally includes all or part of any suitable peptidecomprising a desired biological activity/function. Indeed, in someaspects, a TFPI-inhibitory peptide is operably linked to, for instance,one or more of the following: a peptide with long circulating half life,a marker protein, a peptide that facilitates purification of theTFPI-inhibitory peptide, a peptide sequence that promotes formation ofmultimeric proteins, or a fragment of any of the foregoing. Suitablefusion partners include, but are not limited to, a His tag, a FLAG tag,a strep tag, and a myc tag. Optionally, the TFPI-inhibitor peptide isfused to one or more entities that enhance the half life of the peptide.Half life can be increased by, e.g., increasing the molecular weight ofthe TFPI-binding peptide to avoid renal clearance and/or incorporating aligand for the nFc receptor-mediated recycling pathway. In oneembodiment, the TFPI-binding peptide is fused to or chemicallyconjugated to (as described further below) an albumin polypeptide or afragment thereof (e.g., human serum albumin (HSA) or bovine serumalbumin (BSA)). The albumin fragment comprises 10%, 25%, 50%, or 75% ofthe full length albumin protein. Alternatively or in addition, theTFPI-binding peptide comprises an albumin binding domain or fatty acidthat binds albumin when administered in vivo. Other suitable fusionpartners include, but are not limited to, a proline-alanine-serinemultimer (PASylation) and an antibody or fragment thereof (e.g., an Fcportion of an antibody).

In one embodiment, two or more TFPI-inhibitory peptides are fusedtogether, linked by a multimerization domain, or attached via chemicallinkage to generate a TFPI-inhibitory peptide complex. TheTFPI-inhibitor peptides may be the same or different. Thus, theinvention provides a homo-dimer (i.e., a dimer comprising two identicalTFPI-binding peptides), a homo-multimer (i.e., a complex comprisingthree or more identical TFPI-binding peptides), a hetero-dimer (i.e., adimer comprising two different TFPI-binding peptides), andheteromultimer (i.e., a complex comprising three or more TFPI-bindingpeptides, wherein at least two of the TFPI-binding peptides aredifferent) comprising or consisting of any of the peptides describedherein, optionally attached by one or more linkers. An exemplaryTFPI-binding peptide dimer is JBT2496 (SEQ ID NO: 4211)

“Derivatives” are included in the invention and include TFPI-inhibitorypeptides that have been chemically modified in some manner distinct fromaddition, deletion, or substitution of amino acids. In this regard, apeptide of the invention provided herein is chemically bonded withpolymers, lipids, other organic moieties, and/or inorganic moieties.Examples of peptide and protein modifications are given in Hermanson,Bioconjugate Techniques, Academic Press, (1996). The TFPI-bindingpeptides described herein optionally comprise a functional group thatfacilitates conjugation to another moiety (e.g., a peptide moiety).Exemplary functional groups include, but are not limited to,isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride,aldehyde, epoxide, oxirane, carbonate, arylating agent, imidoester,carbodiimide, anhydride, alkyl halide derivatives (e.g., haloacetylderivatives), maleimide, aziridine, acryloyl derivatives, arylatingagents, thiol-disulfide exchange reagents (e.g., pyridyl disulfides orTNB thiol), diazoalkane, carboyldiimadazole, N,N′-Disuccinyl carbonate,N-Hydroxysuccinimidyl chloroformate, and hydrazine derivatives.Maleimide is useful, for example, for generating a TFPI-binding peptidethat binds with albumin in vivo.

Derivatives are prepared in some situations to increase solubility,stability, absorption, or circulating half life. Various chemicalmodifications eliminate or attenuate any undesirable side effect of theagent. In one aspect, the invention includes TFPI-binding peptidescovalently modified to include one or more water soluble polymerattachments. A water soluble polymer (or other chemical moiety) isattached to any amino acid residue, although attachment to the N- orC-terminus is preferred in some embodiments. Optionally, a polymer isattached to the peptide via one or more amino acids or building blocksthat offer functional groups that facilitate polymer attachment. Forexample, JBT2315 comprises a C-terminal cysteine (position X4021 withrespect to formula (XI)), which facilitates the addition of, e.g., amaleimide polyethylene glycol (PEG). Useful polymers include, but arenot limited to, PEG (e.g., PEG approximately 40 kD, 30 kD, 20 kD, 10,kD, 5 kD, or 1 kD in size), polyoxyethylene glycol, polypropyleneglycol, monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch,cellulose, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propyleneglycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer,polysialic acid (PSA), polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol, as well as mixtures of any of the foregoing. In oneaspect, the peptide of the invention is a PEGylated peptide. PEGmoieties are available in different shapes, e.g., linear or branched.For further discussion of water soluble polymer attachments, see U.S.Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and4,179,337. Other moieties useful for improving peptide half life orstability are described herein and include, for instance, albumin(optionally modified to allow conjugation to the inventive peptide),fatty acid chains (e.g., C12-C18 fatty acid, such as a C14 fatty acid),an antibody or fragment thereof (e.g., an Fc portion of an antibody),and proline-alanine-serine multimers.

In another aspect, a peptide derivative includes a targeting moietyspecific for a particular cell type, tissue, and/or organ.Alternatively, the peptide is linked to one or more chemical moietiesthat facilitate purification, detection, multimerization, binding withan interaction partner, and characterization of peptide activity. Anexemplary chemical moiety is biotin. Other moieties suitable forconjugation to the TFPI-binding peptide of the invention include, butare not limited to, a photosensitizer, a dye, a fluorescence dye, aradionuclide, a radionuclide-containing complex, an enzyme, a toxin, anda cytotoxic agent. Photosensitizers include, e.g., Photofrin, Visudyne,Levulan, Foscan, Metvix, Hexvix®, Cysview™, Laserphyrin, Antrin,Photochlor, Photosens, Photrex, Lumacan, Cevira, Visonac, BF-200 ALA,and Amphinex. If desired, a His tag, a FLAG tag, a strep tag, or a myctag is conjugated to the peptide.

In addition, in one aspect, the peptides of the invention are acylatedat the N-terminal amino acid of the peptide. In another aspect, thepeptides of the invention are amidated at the C-terminal amino acid ofthe peptide. In a still further aspect, the peptides of the inventionare acylated at the N-terminal amino acid of the peptide and areamidated at the C-terminal amino acid of the peptide.

Derivatives also include peptides comprising modified ornon-proteinogenic amino acids or a modified linker group (see, e.g.,Grant, Synthetic Peptides: A User's Guide, Oxford University Press(1992)). Modified amino acids include, for example, amino acids whereinthe amino and/or carboxyl group is replaced by another group.Non-limiting examples include modified amino acids incorporatingthioamides, ureas, thioureas, acylhydrazides, esters, olefines,sulfonamides, phosphoric acid amides, ketones, alcohols, boronic acidamides, benzodiazepines and other aromatic or non-aromatic heterocycles(see Estiarte et al., Burgers Medicinal Chemistry, 6th edition, Volume1, Part 4, John Wiley & Sons, New York (2002)). Modified amino acids areoften connected to the peptide with at least one of the above mentionedfunctional groups instead of an amide bond. Non-proteinogenic aminoacids include, but are not limited, to β-alanine (Bal), norvaline (Nva),norleucine (Nle), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyric acid(Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (Orn), hydroxyproline(Hyp), taurine, sarcosine, citrulline (Cit), cysteic acid (Coh),cyclohexylalanine (Cha), methioninesulfoxide (Meo), methioninesulfone(Moo), homoserinemethylester (Hsm), propargylglycine (Eag),5-fluorotryptophan (5Fw), 6-fluorotryptophan (6Fw),3′,4′-dimethoxyphenyl-alanine (Ear), 3′,4′-difluorophenylalanine (Dff),4′-fluorophenyl-alanine (Pff), 1-naphthyl-alanine (1Ni),1-methyltryptophan (1Mw), penicillamine (Pen), homoserine (Hse),t-butylglycine, t-butylalanine, phenylglycine (Phg), benzothienylalanine(Bta), L-homo-cysteine (Hcy), N-methyl-phenylalanine (Nmf),2-thienylalanine (Thi), 3,3-diphenylalanine (Ebw), homophenylalanine(Hfe) and S-benzyl-L-cysteine (Ece). The structures of many of thenon-proteinogenic amino acids are provided in Table 2. These and othernon-proteinogenic amino acids may exist as D- or L-isomers. Examples ofmodified linkers include, but are not limited to, the flexible linker4,7,10-trioxa-1,13-tridecanediamine (Ttds), glycine, 6-aminohexanoicacid, beta-alanine (Bal), pentynoic acid (Pyn), and combinations ofTtds, glycine, 6-aminohexanoic acid and Bal.

Homologs of the amino acids constituting the peptides of the inventionmay be as set forth in Table 3. In any embodiment, one or more aminoacids of the TFPI-binding peptide are substituted with a homolog.

TABLE 3 Amino Acid Exemplary homologs A Aib, Bal, Eag, Nma, Abu, G, M,Nva, Nle C S, A, Hcy, M, L, I, V, Nmc, β-Cysteine D E, Homoglutamicacid, γ-Hydroxy-glutamic acid, γ-Carboxy- glutamic acid, Nmd, β-Asparticacid, N, Q, Cysteic acid E D, Glu, Homoglutamic acid, γ-Hydroxy-glutamicacid, γ-Carboxy-glutamic acid, α-Aminoadipic acid, Nme, β-glutamic acid,Q, N, Cysteic acid F Hfe, Nmf, β-Phenylalanine, Phg, Bhf,Thienylalanine, Benzothienylalanine, Bromophenylalanine,Iodophenylalanione, Chlorophenylalanine, Methylphenylalanine,Nitrophenylalanine, Y, W, Naphtylalanine, Tic G A, Nmg H Nmh,1-Methylhistidine, 3-Methylhistidine, Thienylalanine I L, V, Hle, Nva,Nle, β-Isoleucine, Nml, M, Nmi K Nmk, R, Nmr, β-Lysine, Dab, Dap,β-(1-Piperazinyl)-alanine, 2,6-Diamino-4-hexynoic acid,delta-Hydroxy-lysine, Har, omega-Hydroxy-norarginine,omega-Amino-arginine, omega-Methyl-arginine, β-(2-Pyridyl)-alanine,β-(3- Pyridyl)-alanine, 3-Amino-tyrosine, 4-Amino-phenylalanine, Hci,Cit L I, V, Hle, Nle, Nva, β-Isoleucine, Nml, M M I, V, Hle, Nva, R,Har, Nmm, Methioninesulfone N Nmn, β-Asparagine, Q, Nmq, β-Glutamine,Cys(3-propionic acid amide)-OH, Cys(O2-3-propionic acid amide)-OH PAzetidine-2-carboxylic acid, Hyp, α-Methyl-methionine, 4-Hydroxy-piperidine-2-carboxylic acid, Pip, α-Methyl-Pro Q N, Nmn, Nmq,β-Glutamine, Cys(3-propionic acid amide)-OH, Cys(O2-3-propionic acidamide)-OH R Nmk, K, Nmr, β-Lysine, Dab, Dap, Orn, β-(1-Piperazinyl)-alanine, 2,6-Diamino-4-hexynoic acid, delta-Hydroxy-lysine, Har,omega-Hydroxy-norarginine, omega-Amino-arginine, omega-Methyl-arginine,β-(2-Pyridyl)-alanine, β-(3- Pyridyl)-alanine, 3-Amino-tyrosine,4-Amino-phenylalanine, Hci, Cit, Hle, L, Nle, M S T, Hse, β-Serine, C,β-Cyano-alanine, allo-Threonine T S, Homothreonine, β-Threonine,allo-Threonine V L, I, Hle, Nva, Nle, β-Valine, Nmv, M, Nmi, Nml W Nmw,β-Tryptophan, F, Hfe, Nmf, β-Phenylalanine, Phg, Bhf, Thienylalanine,Benzothienylalanine, Bromophenylalanine, Iodophenylalanine,Chlorophenylalanine, Methylphenylalanine, Nitrophenylalanine, Y,Naphtylalanine, Tic Y Nmy, β-Tyrosine,, F, Hfe, Nmf, β-Phenylalanine,Phg, Bhf, Thienylalanine, Benzothienylalanine, Bromophenylalanine,Iodophenylalanine, Chlorophenylalanine, Methylphenylalanine,Nitrophenylalanine, W, Naphtylalanine, Tic

Derivatives also include peptides comprising amino acids having modifiedsubstituents, such as amino acids modified by halogenation with, e.g.,fluorine, chlorine, iodine, or bromine. In some embodiments, theTFPI-binding peptide comprises a halogenated aromatic amino acid, suchas phenylalanine.

In some embodiments, the peptide (CO—NH) linkages joining amino acidswithin the peptide of the invention are reversed to create a“retro-modified” peptide, i.e., a peptide comprising amino acid residuesassembled in the opposite direction (NH—CO bonds) compared to thereference peptide. The retro-modified peptide comprises the same aminoacid chirality as the reference peptide. An “inverso-modified” peptideis a peptide of the invention comprising amino acid residues assembledin the same direction as a reference peptide, but the chirality of theamino acids is inverted. Thus, where the reference peptide comprisesL-amino acids, the “inverso-modified” peptide comprises D-amino acids,and vice versa. Inverso-modified peptides comprise CO—NH peptide bonds.A “retro-inverso modified” peptide refers to a peptide comprising aminoacid residues assembled in the opposite direction and which haveinverted chirality. A retro-inverso analogue has reversed termini andreversed direction of peptide bonds (i.e., NH—CO), while approximatelymaintaining the side chain topology found in the reference peptide.Retro-inverso peptidomimetics are made using standard methods, includingthe methods described in Meziere et al, J. Immunol., 159, 3230-3237(1997), incorporated herein by reference. Partial retro-inverso peptidesare peptides in which only part of the amino acid sequence is reversedand replaced with enantiomeric amino acid residues.

TFPI-binding peptides of the invention (including TFPI inhibitorpeptides) are made in a variety of ways. In one aspect, the peptides aresynthesized by solid phase synthesis techniques including thosedescribed in Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Davis etal., Biochem. Intl., 10, 394-414 (1985); Larsen et al., J. Am. Chem.Soc., 115, 6247 (1993); Smith et al., J. Peptide Protein Res., 44, 183(1994); O'Donnell et al., J. Am. Chem. Soc., 118, 6070 (1996); Stewartand Young, Solid Phase Peptide Synthesis, Freeman (1969); Finn et al.,The Proteins, 3rd ed., vol. 2, pp. 105-253 (1976); and Erickson et al.,The Proteins, 3rd ed., vol. 2, pp. 257-527 (1976). Alternatively, theTFPI-binding peptide (e.g., the TFPI-inhibitory peptide) is expressedrecombinantly by introducing a nucleic acid encoding a TFPI-bindingpeptide (e.g., a TFPI-inhibitory peptide) into host cells, which arecultured to express the peptide. Such peptides are purified from thecell culture using standard protein purification techniques.

The invention also encompasses a nucleic acid comprising a nucleic acidsequence encoding a TFPI-inhibitory peptide of the invention. Methods ofpreparing DNA and/or RNA molecules are well known in the art. In oneaspect, a DNA/RNA molecule encoding a peptide provided herein isgenerated using chemical synthesis techniques and/or using polymerasechain reaction (PCR). If desired, a TFPI-inhibitory peptide codingsequence is incorporated into an expression vector. One of ordinaryskill in the art will appreciate that any of a number of expressionvectors known in the art are suitable in the context of the invention,such as, but not limited to, plasmids, plasmid-liposome complexes, andviral vectors. Any of these expression vectors are prepared usingstandard recombinant DNA techniques described in, e.g., Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994). Optionally, the nucleic acid isoperably linked to one or more regulatory sequences, such as a promoter,activator, enhancer, cap signal, polyadenylation signal, or other signalinvolved with the control of transcription or translation.

Any of the TFPI-inhibitory peptides of the invention or nucleic acidsencoding the peptides also is provided in a composition (e.g., apharmaceutical composition). In this regard, the peptide is formulatedwith a physiologically-acceptable (i.e., pharmacologically-acceptable)carrier, buffer, excipient, or diluent, as described further herein.Optionally, the peptide is in the form of a physiologically acceptablesalt, which is encompassed by the invention. “Physiologically acceptablesalts” means any salts that are pharmaceutically acceptable. Someexamples of appropriate salts include acetate, hydrochloride,hydrobromide, sulfate, citrate, tartrate, glycolate, and oxalate. Ifdesired, the composition comprises one or more additionalpharmaceutically-effective agents.

The peptide provided herein optionally inhibits at least one TFPI-1(e.g., TFPI-1α or TFPI-1β) activity such as, but not limited to, anactivity that downregulates the blood coagulation cascade. Without beingbound by any specific mechanism of action, a proposed mechanism ofinhibition may involve preventing formation of the quaternaryTF-FVIIA-FXA-TFPI complex. The peptide may inhibit binding(competitively or allosterically) of TFPI to FXa (e.g., inhibit bindingof TFPI Kunitz domain 2 to Factor Xa or interrupt binding of TFPI Kunitzdomain 1 to an exosite of Factor Xa), the TF/FVIIa complex (e.g.,inhibit binding of TFPI Kunitz domain 1 to the TF/FVIIa complex), TFalone, and/or FVIIa alone. With TFPI activity diminished, TF and FVIIaare free to activate FX which, in turn, enhances conversion ofprothrombin to thrombin. Surprisingly, in one embodiment, the peptide ofthe invention that binds Kunitz domain 1 interferes with TFPI-mediatedinhibition of FXa. Thus, the invention provides a method of, e.g.,inhibiting TFPI-mediated downregulation of the extrinsic and/or commonpathway of the coagulation cascade and/or enhancing FXa-mediatedconversion of prothrombin to thrombin, by administering to a subject apeptide described herein that binds Kunitz domain 1.

In one aspect, the peptide of the invention exhibits TFPI antagonisticactivity in model and/or plasmatic systems. An exemplary model systemfor determining TFPI-inhibitory activity is the extrinsic tenase assay,which tests the ability of candidate peptides to restore extrinsiccomplex-mediated FX activation in the presence of TFPI (which is anatural inhibitor of the FX activation reaction) (see, e.g., Lindhout etal., Thromb. Haemost., 74, 910-915 (1995)). Another model system forcharacterizing TFPI-inhibitory activity is the FXa inhibition assay,wherein FXa activity is measured in the presence of TFPI (see Sprecheret al., PNAS, 91, 3353-3357 (1994)). The extrinsic tenase assay and theFXa inhibition assay are further described in Example 3. Optionally, thepeptide of the invention enhances FX activation in the presence of TFPIwith a half maximal effective concentration (EC₅₀) of less than or equalto 1×10⁻⁴M, less than or equal to 1×10⁻⁵M, less than or equal to1×10⁻⁶M, or less than or equal to 1×10⁻⁷M.

In one aspect, TFPI-antagonist activity is characterized in aplasma-based assay. Thrombin formation is triggered in plasmasubstantially lacking FVIII or FIX activity (e.g., the residualcoagulation factor activity is lower than 1%) in the presence of acandidate peptide. Thrombin formation can be detected using afluorogenic or chromogenic substrate, as described in Example 4. Asystem for measuring thrombin activity is provided by Thrombinoscope BV(Maastricht, The Netherlands). Prothrombin conversion is measured using,e.g., a Thrombograph™ (Thermo Scientific, Waltham, Mass.), and theresulting data is compiled into a Calibrated Automatic Thrombogramgenerated by Thrombinoscope™ software available from Thrombinoscope BV.In certain embodiments, the TFPI-inhibitory peptide increases the amountof peak thrombin generated during the assay and/or decreases the timerequired to achieve peak thrombin formation. For example, the peptideimproves TFPI-regulated thrombin generation in the absence of FVIII(e.g., in FVIII-depleted plasma) to at least 1% of the level ofTFPI-dependent thrombin generation in normal plasma. Generally, normal(unafflicted) plasma contains about 0.5 U/mL to about 2 U/mL FactorVIII. Accordingly, in some instances, a TFPI-inhibitor peptide willenhance thrombin formation in the absence of FVIII to at least about 1%of that observed in the presence of 0.5 U/mL to 2 U/mL FVIII. In furtherembodiments, the peptide enhances thrombin formation in the absence ofFactor VIII to at least about 2%, at least about 3%, at least about 5%,at least about 7%, or at least about 10% of the level of thrombinformation in normal plasma, i.e., in the presence of physiologicallevels of Factor VIII. In various aspects, the peptide is administeredto an animal model of thrombin deficiency or hemophilia to characterizeTFPI inhibitory activity in vivo. Such in vivo models are known in theart and include for example, mice administered anti-FVIII antibodies toinduce hemophilia A (Tranholm et al., Blood, 102, 3615-3620 (2003));coagulation factor knock-out models such as, but not limited to, FVIIIknock-out mice (Bi et al., Nat. Genet., 10(1), 119-121 (1995)) and FIXknock-out mice (Wang et al., PNAS, 94(21), 11563-66 (1997)); inducedhemophilia-A in rabbits (Shen et al., Blood, 42(4), 509-521 (1973)); andChapel Hill HA dogs (Lozier et al., PNAS, 99, 12991-12996 (2002)).

Various peptides bind TFPI from any source including, but not limitedto, mouse, rat, rabbit, dog, cat, cow, horse, pig, guinea pig, andprimate. In one embodiment, the peptide binds human TFPI. Optionally,the TFPI-inhibitory peptide binds TFPI from more than one species (i.e.,the peptide is cross-reactive among multiple species). In certainaspects, the peptide binds TFPI with a dissociation constant (K_(D)) ofless than or equal to 1×10⁻⁴M, less than or equal to 1×10⁻⁵M, less thanor equal to 1×10⁻⁶M, or less than or equal to 1×10⁻⁷ M. Affinity may bedetermined using, for example and without limitation, any one, two, ormore of a variety of techniques, such as affinity ELISA assay, acompetitive ELISA assay, and/or surface plasmon resonance (BIAcore™)assay. When characterized using a competitive (IC₅₀) ELISA assay, thepeptide of the invention optionally demonstrates an IC₅₀ of less than orequal to about 50,000 nM. For example, the peptide demonstrates an IC₅₀of less than or equal to about 10,000 nM, such as an IC₅₀ of less thanor equal to about 5,000 nM, less than or equal to about 1,000 nM, orless than or equal to about 500 nM. In one aspect, the peptidedemonstrates an IC₅₀ of less than or equal to about 250 nM, less than orequal to about 100 nM, less than or equal to about 50 nM, or less thanor equal to about 10 nM. Exemplary peptides and their IC₅₀ values areprovided in FIGS. 32A-32AM, 33, 34A-34J, 35, 36A-36Q, 37, 38A-38B, and39; in some instances, the peptides are classified into Groups A, B, C,D, E, F, and G (see Table 4 in Example 1) based on their IC₅₀ values. Invarious aspects, the invention provides peptides falling within GroupsA, B, C, D, E, F, and/or G as defined in Table 4. Affinity may also bedetermined by a kinetic method or an equilibrium/solution method. Suchmethods are described in further detail herein or known in the art.

Another suitable assay for characterizing the inventive peptides is ak_(off) assay, which examines a peptide's release from TFPI. The k_(off)assay result is not the dissociation rate constant, but a percentage ofcompetitor peptide blocked from TFPI binding by a test peptide after anincubation period with TFPI. An exemplary k_(off) assay includes thefollowing steps: 1) incubation of a TFPI-coated microtiter plate with anamount of test peptide resulting in approximately 90% TFPI occupation;2) removal of unbound test peptide; 3) addition of a biotinylated tracer(i.e., competitor) peptide that competes with the test peptide forbinding to TFPI; 4) incubation for a period of time during which bindingsites released by the test peptide is occupied by the tracer; 5) removalof unbound tracer and test peptide; and 6) detection of bound tracer bya chromogenic reaction using streptavidin-horseradish peroxidaseconjugate. The resulting signal is indicative of binding sites freed bythe test peptide. A test peptide that does not dissociate from TFPIduring the incubation period yields a weaker signal compared to ananalyte that dissociates completely.

As with all binding agents and binding assays, one of skill in the artrecognizes that the various moieties to which a binding agent should notdetectably bind in order to be biologically (e.g., therapeutically)effective would be exhaustive and impractical to list. Therefore, theterm “specifically binds” refers to the ability of a peptide to bindTFPI with greater affinity than it binds to an unrelated control proteinthat is not TFPI. For example, the peptide may bind to TFPI with anaffinity that is at least, 5, 10, 15, 25, 50, 100, 250, 500, 1000, or10,000 times greater than the affinity for a control protein. In someembodiments, the peptide binds TFPI with greater affinity than it bindsto an “anti-target,” a protein or other naturally occurring substance inhumans to which binding of the peptide might lead to adverse effects.Several classes of peptides or proteins are potential anti-targets.Because TFPI-inhibitory peptides exert their activity in the bloodstream and/or at the endothelium, plasma proteins represent potentialanti-targets. Proteins containing Kunitz domains (KDs) are potentialanti-targets because KDs of different proteins share a significantsimilarity. Tissue Factor Pathway Inhibitor-2 (TFPI-2) is highly similarto TFPI-1α and, like TFPI-1α, contains KDs (Sprecher et al., PNAS, 91,3353-3357 (1994)). Thus, in one aspect, the peptide of the inventionbinds to TFPI with an affinity that is at least 5, 10, 15, 25, or 50times greater than the affinity for an anti-target, such as TFPI-2.

Optionally, the TFPI-binding peptide demonstrates one or more desiredcharacteristics described herein, and the amino acid sequence of apeptide can be modified to optimize binding, stability, and/or activity,if desired. An exemplary TFPI-binding peptide binds TFPI with a K_(D) ofless than or equal to 20 nM and/or exhibits a binding affinity for TFPIthat is at least 100 times greater than the binding affinity for ananti-target. Alternatively or in addition, the TFPI-binding peptideenhances FX activation in the presence of TFPI with an EC₅₀ (as measuredusing any suitable assay, such as the assays described here) of lessthan or equal to 50 nM and/or enhances thrombin formation in the absenceof Factor VIII to at least about 20% (e.g., 40%) of the level ofthrombin formation in plasma containing physiological levels of FactorVIII. Alternatively or in addition, the TFPI-binding peptide achieves adesired level of plasma stability (e.g., 50% or more of a dose remainsin plasma after 12 hours) and/or demonstrates a desired half life invivo (e.g., at least two, three, four, five, six, seven, eight, nine, orten hours). Alternatively or in addition, the TFPI-binding peptideexhibits a desired level of bioavailability, such as a desired level ofbioavailability following subcutaneous administration (e.g., greaterthan or equal to 5%, 10%, 15%, 20%, 25%, 30%, or 50%) and/ordemonstrates a desired level of TFPI-inhibitory activity at a given dosein vivo.

The invention further includes a method of inhibiting TFPI-1. The methodcomprises contacting TFPI with a TFPI-binding peptide as describedherein. Any degree of TFPI-activity inhibition is contemplated. Forexample, a TFPI-inhibitory peptide reduces TFPI-inhibition of theextrinsic pathway at least about 5% (e.g., at least about 10%, at leastabout 25%, or at least about 30%). In some embodiments, theTFPI-inhibitory peptide reduces TFPI activity within the extrinsicpathway at least about 50%, at least about 75%, or at least about 90%compared to TFPI activity in the absence of the peptide.

In one aspect of the invention, TFPI-binding peptides are used to detectand/or quantify TFPI in vivo or in vitro. An exemplary method ofdetecting and/or quantifying TFPI in a sample comprises (a) contacting asample with a TFPI-binding peptide of the invention, and (b) detectingbinding of the TFPI-binding peptide to TFPI.

The invention further includes a method for targeting biologicalstructures (including, but not limited to, cell surfaces and endotheliallining) where TFPI is located. The method comprises contacting thebiological structure (e.g., including, without limitation, a celldisplaying TFPI on the cell surface) with a TFPI-binding peptidedescribed herein, optionally conjugated to a moiety that adds additionalfunctionality to the peptide. The moiety can be a dye (such as afluorescence dye), a radionuclide or a radionuclide-containing complex,a protein (e.g., an enzyme, a toxin, or an antibody) or a cytotoxicagent. For example, the peptide is linked or conjugated to an effectormoiety that facilitates peptide detection and/or purification and/orcomprises therapeutic properties. In one aspect, the TFPI-bindingpeptide or peptide conjugate is administered to a mammal to target aTFPI-displaying cell within the mammal. Optionally, the method furthercomprises detecting binding of the TFPI-binding peptide to TFPI. Themethod is useful for therapy and diagnosis of disease where TFPI is asuitable diagnostic marker or TFPI-expressing cells are a target for atherapeutic approach.

Peptide-TFPI complexes are directly or indirectly detected. Detectionmoieties are widely used in the art to identify biological substancesand include, for example, dye (e.g., fluorescent dye), radionuclides andradionuclide-containing complexes, and enzymes. In some aspects,peptide-TFPI binding is detected indirectly. In this regard, the peptideis optionally contacted with an interaction partner that binds thepeptide of invention without significantly interfering with peptide-TFPIbinding, and the interaction partner is detected. Exemplary interactionpartners include, but are not limited to, antibodies, antigen-bindingantibody fragments, anticalins and antibody mimetics, aptamers,streptavidin, avidin, neutravidin, and spiegelmers. Optionally, theinteraction partner comprises a detection moiety to facilitate detectionof an interaction partner-peptide complex. The TFPI-binding peptide is,in some embodiments, modified to facilitate binding of an interactionpartner. For example, in one aspect, the TFPI-binding peptide isconjugated to biotin, which is bound by an interaction partnercomprising streptavidin. An exemplary interaction partner comprisesstrepavidin fused to horseradish peroxidase, which is detected in, e.g.,an ELISA-like assay. Alternatively, the TFPI-binding peptide is modifiedto include an antibody epitope, and binding of the correspondingantibody to the peptide-TFPI complex is detected. Methods of detecting,e.g., antibodies and fragments thereof, are well understood in the art.

Peptide-TFPI complexes and interaction partner-peptide complexes areidentified using any of a number of methods, such as, but not limitedto, biochemical assays (e.g., enzymatic assays), spectroscopy (e.g.,detection based on optical density, fluorescence, FRET, BRET, TR-FRET,fluorescence polarization, electrochemoluminescence, or NMR), positronemission tomography (PET), and single Photon Emission ComputedTomography (SPECT). Detectable moieties that facilitate fluorescencedetection of peptide-TFPI complexes or interaction partner-peptidecomplexes include, but are not limited to, fluorescein, Alexa Fluor®350, Marina Blue™, Cascade Yellow™, Alexa Fluor® 405, Pacific Blue™,Pacific Orange™, Alexa Fluor® 430, Alexa Fluor® 488, Oregon Green® 488,Alexa Fluor® 500, Oregon Green® 514, Alexa Fluor® 514, Alexa Fluor® 532,Alexa Fluor® 555, Tetramethylrhodamine, Alexa Fluor® 546, Rhodamine B,Rhodamine Red™-X, Alexa Fluor® 568, Alexa Fluor® 594, Texas Red®, TexasRed®-X, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, AlexaFluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, AlexaFluor® 750, B-Phycoerythrin, R-Phycoerythrin, Allophycocyanin, BODIPY®,Cy3, Cy5, TAMRA, and fluorescent proteins (GFP and derivatives thereof).An example of a TFPI-binding peptide comprising a fluorescent detectionmoiety is JBT2454 (FAM-Ttds-FQSKpNVHVDGYFERL-Aib-AKL-NH2 (SEQ ID NO:4171)), which is labeled with 5,6-carboxyfluoresceine.

Radioactive labels also are used to detect biological materials (e.g.,TFPI, TFPI-binding peptides, or TFPI-binding peptide-TFPI complexes),and, in some instances, are attached to peptides or interaction partnersusing a chelator, such as (but not limited to) EDTA (ethylene diaminetetra-acetic acid), DTPA (diethylene triamine pentaacetic acid), CDTA(cyclohexyl 1,2-diamine tetra-acetic acid), EGTA(ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic), HBED(N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid), TTHA(triethylene tetramine hexa-acetic acid), DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetra-acetic acid), HEDTA(hydroxyethyldiamine triacetic acid), or TETA(1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N″′-tetra-acetic acid).Examples of radioactive labels include ^(99m)Tc, ²⁰³Pb, ⁶⁶Ga, ⁶⁷Ga,⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ^(114m)In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe,^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ^(117m)Sn, ¹²¹Sn,¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh and ¹¹¹Ag.Paramagnetic metals also are detectable moieties that are suitable forattachment to TFPI-binding peptides or interaction partners, optionallyvia chelator complex. Examples of paramagnetic metals include, forexample, Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho, and Er.

TFPI-binding peptides, themselves, are, in some aspects, modified toinclude one or more amino acids with detectable substituents ornuclides. In this regard, in one embodiment, the TFPI-binding peptidecomprises at least one amino acid comprising a detectable isotope (e.g.,13C, 14C, 35S, 3H, 18O or 15N), and/or an amino acid that is halogenatedwith, e.g., ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br or ⁸²Br. Aminoacids suitable for halogenation include, but are not limited to,tyrosine and tryptophan.

The invention also provides a method for diagnosing a subject sufferingfrom a disease or disorder, or at risk of suffering from a disease ordisorder, wherein the disease or disorder is associated with or causedby aberrant TFPI activity. The method comprises administering to thesubject the TFPI-binding peptide and detecting the TFPI-peptide complex.In some instances, the peptide is conjugated to a detectable moiety, andthe method comprises detecting the detectable moiety. Exemplarydetectable moieties are described herein. In other instances, the methodcomprises administering to the subject a TFPI-binding peptideinteraction partner that binds the TFPI-binding peptide, and detectingthe interaction partner. If desired, the interaction partner comprisesor is conjugated to a detectable moiety, and the detectable moiety isdetected. The presence of the detectable moiety indicates the presenceof TFPI, thereby allowing diagnosis of a disease or disorder associatedwith TFPI (e.g., a disease or disorder which (i) can be treated byinhibiting TFPI or (ii) comprises symptoms which can be ameliorated orprevented by inhibiting TFPI). If administration of the peptide to thesubject is not desired, a biological sample is obtained from thesubject, contacted with the TFPI-binding peptide as described herein,and TFPI-peptide complexes are detected.

The peptides of the invention bind TFPI and, therefore, are useful forpurifying TFPI or recombinant TFPI from a biological sample (e.g., abiological fluid, such as serum), fermentation extract, tissuepreparations, culture medium, and the like. The invention includesmethods of using the TFPI-binding peptide in the commercial productionof TFPI or in a method of characterizing TFPI molecules. For example,the invention includes a method of purifying TFPI. The method comprisescontacting a sample containing TFPI with a peptide as defined hereinunder conditions appropriate to form a complex between TFPI and thepeptide; removing the complex from the sample; and, optionally,dissociating the complex to release TFPI. Exemplary conditionsappropriate to form a complex between TFPI and the peptide are disclosedin the Examples, and such conditions can be easily modified todissociate the TFPI-peptide complex. In some embodiments, the peptide isimmobilized to a support, e.g., a solid support, to facilitate recoveryof TFPI. For example, in one embodiment, the peptide is immobilized tochromatography stationary phase (e.g., silica, affinity chromatographybeads, or chromatography resins), a sample comprising TFPI is applied tothe stationary phase such that TFPI-peptide complexes are formed, theremainder of the sample is removed from the stationary phase, and TFPIis eluted from the stationary phase. In this regard, the peptides of theinvention are, in one aspect, suitable for use in affinitychromatography techniques.

A method of enhancing thrombin formation in a clotting factor-deficientsubject also is provided. The method comprises administering to thesubject a peptide provided herein under conditions effective to inhibitTFPI. In this regard, the TFPI-inhibitory peptide is administered in anamount and under conditions effective to enhance thrombin formation inthe subject. By “clotting factor-deficient” is meant that the subjectsuffers from a deficiency in one or more blood factors required forthrombin formation, such as FVIII, FIX, or FXI. Indeed, in oneembodiment, the subject is deficient in FVIII. Alternatively or inaddition, the subject is deficient in Factor IX. Clotting factordeficiencies are identified by examining the amount of factor in aclinical sample. Practitioners classify hemophilia according to themagnitude of clotting factor deficiency. Subjects suffering from mildhemophilia have approximately 5% to 30% of the normal amount (1 U/ml) ofFactor VIII or Factor IX. Moderate hemophilia is characterized byapproximately 1% to 5% of normal Factor VIII, Factor IX, or Factor XIlevels, while subjects suffering from severe hemophilia have less than1% of the normal amount of Factor VIII, Factor IX, or Factor XI.Deficiencies can be identified indirectly by activated partialthromboplastin time (APTT) testing. APTT testing measures the length oftime required for a blood clot to form, which is longer for patientswith Factor VIII Deficiency (hemophilia A), Factor IX Deficiency(hemophilia B), and Factor XI Deficiency (hemophilia C) compared topatients with normal clotting factor levels. Almost 100% of patientswith severe and moderate Factor VIII deficiency can be diagnosed with anAPTT. The invention further includes enhancing thrombin formation in asubject that does not suffer from a clotting factor deficiency. Themethod comprises administering to a subject (e.g., a subject comprisingnormal, physiological levels of clotting factor) a peptide providedherein under conditions effective to enhance thrombin formation.

In one aspect, the TFPI-inhibitory peptide is used for increasing bloodclot formation in a subject. The method of increasing blood clotformation comprises administering to the subject a peptide describedherein in an amount and under conditions effective to increase bloodclot formation. It will be appreciated that the method need notcompletely restore the coagulation cascade to achieve a beneficial(e.g., therapeutic) effect. Any enhancement or increase in thrombin orblood clot formation that reduces the onset or severity of symptomsassociated with clotting factor deficiencies is contemplated. Methods ofdetermining the efficacy of the method in promoting thrombin formationand blood clotting are known in the art and described herein.

The invention further includes a method of treating a blood coagulationdisorder in a subject, the method comprising administering to thesubject one or more TFPI-inhibitory peptides, such as any one or more ofthe peptides described herein, in an amount and under conditionseffective to treat the blood coagulation disorder in the subject. In oneaspect, the peptide is a recombinant or synthetic peptide that inhibitsTFPI activity. “Coagulation disorders” include bleeding disorders causedby deficient blood coagulation factor activity and deficient plateletactivity. Blood coagulation factors include, but are not limited to,Factor V (FV), FVII, FVIII, FIX, FX, FXI, FXIII, FII (responsible forhypoprothrombinemia), and von Willebrand's factor. Factor deficienciesare caused by, for instance, a shortened in vivo-half life of thefactor, altered binding properties of the factor, genetic defects of thefactor, and a reduced plasma concentration of the factor. Coagulationdisorders can be congenital or acquired. Potential genetic defectsinclude deletions, additions and/or substitution within a nucleotidesequence encoding a clotting factor whose absence, presence, and/orsubstitution, respectively, has a negative impact on the clottingfactor's activity. Coagulation disorders also stem from development ofinhibitors or autoimmunity (e.g., antibodies) against clotting factors.In one example, the coagulation disorder is hemophilia A. Alternatively,the coagulation disorder is hemophilia B or hemophilia C.

Platelet disorders are caused by deficient platelet function orabnormally low platelet number in circulation. Low platelet count may bedue to, for instance, underproduction, platelet sequestration, oruncontrolled patent destruction. Thrombocytopenia (plateletdeficiencies) may be present for various reasons, including chemotherapyand other drug therapy, radiation therapy, surgery, accidental bloodloss, and other disease conditions. Exemplary disease conditions thatinvolve thrombocytopenia are: aplastic anemia; idiopathic or immunethrombocytopenia (ITP), including idiopathic thrombocytopenic purpuraassociated with breast cancer; HIV-associated ITP and HIV-relatedthrombotic thrombocytopenic purpura; metastatic tumors which result inthrombocytopenia; systemic lupus erythematosus, including neonatal lupussyndrome splenomegaly; Fanconi's syndrome; vitamin B12 deficiency; folicacid deficiency; May-Hegglin anomaly; Wiskott-Aldrich syndrome; chronicliver disease; myelodysplastic syndrome associated withthrombocytopenia; paroxysmal nocturnal hemoglobinuria; acute profoundthrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmunethrombocytopenia, including maternal alloimmune thrombocytopenia;thrombocytopenia associated with antiphospholipid antibodies andthrombosis; autoimmune thrombocytopenia; drug-induced immunethrombocytopenia, including carboplatin-induced thrombocytopenia andheparin-induced thrombocytopenia; fetal thrombocytopenia; gestationalthrombocytopenia; Hughes' syndrome; lupoid thrombocytopenia; accidentaland/or massive blood loss; myeloproliferative disorders;thrombocytopenia in patients with malignancies; thromboticthrombocytopenia purpura, including thrombotic microangiopathymanifesting as thrombotic thrombocytopenic purpura/hemolytic uremicsyndrome in cancer patients; post-transfusion purpura (PTP); autoimmunehemolytic anemia; occult jejunal diverticulum perforation; pure red cellaplasia; autoimmune thrombocytopenia; nephropathia epidemica;rifampicin-associated acute renal failure; Paris-Trousseauthrombocytopenia; neonatal alloimmune thrombocytopenia; paroxysmalnocturnal hemoglobinuria; hematologic changes in stomach cancer;hemolytic uremic syndromes (e.g., uremic conditions in childhood); andhematologic manifestations related to viral infection includinghepatitis A virus and CMV-associated thrombocytopenia. Plateletdisorders also include, but are not limited to, Von Willebrand Disease,paraneoplastic platelet dysfunction, Glanzman's thrombasthenia, andBernard-Soulier disease. Additional bleeding disorders amenable totreatment with a TFPI-inhibitory peptide include, but are not limitedto, hemorrhagic conditions induced by trauma; a deficiency in one ormore contact factors, such as FXI, FXII, prekallikrein, and highmolecular weight kininogen (HMWK); vitamin K deficiency; a fibrinogendisorder, including afibrinogenemia, hypofibrinogenemia, anddysfibrinogenemia; and alpha2-antiplasmin deficiency. In one embodiment,the TFPI-inhibitory peptide is used to treat excessive bleeding, such asexcessive bleeding caused by surgery, trauma, intracerebral hemorrhage,liver disease, renal disease, thrombocytopenia, platelet dysfunction,hematomas, internal hemorrhage, hemarthroses, hypothermia, menstruation,pregnancy, and Dengue hemorrhagic fever. All of the above are considered“blood coagulation disorders” in the context of the disclosure.

In one aspect, the TFPI-inhibitory peptide of the invention is used toreverse the effects (in whole or in part) of one or more anticoagulantsin a subject. Numerous anticoagulants are known in the art and include,for instance, heparin; coumarin derivatives, such as warfarin ordicumarol; TFPI; AT III; lupus anticoagulant; nematode anticoagulantpeptide (NAPc2); FVIIa inhibitors; active-site blocked FVIIa (FVIIai);active-site blocked FIXa (FIXai); FIXa inhibitors; FXa inhibitors,including fondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906);active-site blocked FXa (FXai); inhibitors of FVa or FVIIIa, includingactivated protein C (APC) and soluble thrombomodulin; thrombininhibitors, including hirudin, bivalirudin, argatroban, andximelagatran; and antibodies or antibody fragments that bind a clottingfactor (e.g., FV, FVII, FVIII, FIX, FX, FXIII, FII, FXI, FXII, vonWillebrand factor, prekallikrein, or high molecular weight kininogen(HMWK)).

As used herein, “treating” and “treatment” refers to any reduction inthe severity and/or onset of symptoms associated with a bloodcoagulation disorder. Accordingly, “treating” and “treatment” includestherapeutic and prophylactic measures. One of ordinary skill in the artwill appreciate that any degree of protection from, or amelioration of,a blood coagulation disorder or symptom associated therewith isbeneficial to a subject, such as a human patient. The quality of life ofa patient is improved by reducing to any degree the severity of symptomsin a subject and/or delaying the appearance of symptoms. Accordingly,the method in one aspect is performed as soon as possible after it hasbeen determined that a subject is at risk for developing a bloodcoagulation disorder (e.g., a deficiency in a clotting factor (e.g.,FVIII, FIX, or FXI) is detected) or as soon as possible after a bloodcoagulation disorder (e.g., hemophilia A, hemophilia B, or hemophilia C)is detected. In an additional aspect, the peptide is administered toprotect, in whole or in part, against excessive blood loss during injuryor surgery.

In view of the above, the invention provides a peptide for use in amethod for the treatment of a subject, such as a method for thetreatment of a disease where the inhibition of TFPI is beneficial. Inone aspect, the disease or disorder is a blood coagulation disorder. Thesubject is suffering from a disease or disorder or is at risk fromsuffering from a disease or disorder (or adverse biological event, suchas excessive blood loss). The method comprises administering to thesubject the peptide of the invention in an amount and under conditionseffective to treat or prevent, in whole or in part, the disease ordisorder. The invention further provides a peptide for use in themanufacture of a medicament. For example, the peptide can be used in themanufacture of a medicament for the treatment of a blood coagulationdisorder, as described in detail herein.

In some embodiments, it is advantageous to administer to a subject anucleic acid comprising a nucleic acid sequence encoding a TFPI-bindingpeptide (e.g., TFPI-inhibitory peptide) of the invention. Such a nucleicacid, in one aspect, is provided instead of, or in addition to, aTFPI-inhibitory peptide. Expression vectors, nucleic acid regulatorysequences, administration methods, and the like, are further describedherein and in U.S. Patent Publication No. 20030045498.

A particular administration regimen for a particular subject willdepend, in part, upon the TFPI-inhibitory peptide of the invention used,the amount of TFPI-binding peptide (e.g., TFPI-inhibitory peptide)administered, the route of administration, the particular ailment beingtreated, considerations relevant to the recipient, and the cause andextent of any side effects. The amount of peptide administered to asubject (e.g., a mammal, such as a human) and the conditions ofadministration (e.g., timing of administration, route of administration,dosage regimen) are sufficient to affect the desired biological responseover a reasonable time frame. Dosage typically depends upon a variety offactors, including the particular TFPI-inhibitory peptide employed, theage and body weight of the subject, as well as the existence andseverity of any disease or disorder in the subject. The size of the dosealso will be determined by the route, timing, and frequency ofadministration. Accordingly, the clinician may titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect, and conventional range-finding techniques are known to those ofordinary skill in the art. Purely by way of illustration, in one aspect,the method comprises administering, e.g., from about 0.1 μg/kg to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 1 μg/kg up to about 75 mg/kg; or5 μg/kg up to about 50 mg/kg; or 10 μg/kg up to about 20 mg/kg. Incertain embodiments, the dose comprises about 0.5 mg/kg to about 20mg/kg (e.g., about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.3 mg/kg, 2.5 mg/kg, 3mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of peptide. Given thechronic nature of many blood coagulation disorders, it is envisionedthat a subject will receive the TFPI-inhibitory peptide over a treatmentcourse lasting weeks, months, or years, and may require one or moredoses daily or weekly. In other embodiments, the TFPI-inhibitory peptideis administered to treat an acute condition (e.g., bleeding caused bysurgery or trauma, or factor inhibitor/autoimmune episodes in subjectsreceiving coagulation replacement therapy) for a relatively shorttreatment period, e.g., one to 14 days.

Suitable methods of administering a physiologically-acceptablecomposition, such as a pharmaceutical composition comprising a peptidedescribed herein, are well known in the art. Although more than oneroute can be used to administer a peptide, a particular route canprovide a more immediate and more effective reaction than another route.Depending on the circumstances, a pharmaceutical composition is appliedor instilled into body cavities, absorbed through the skin or mucousmembranes, ingested, inhaled, and/or introduced into circulation. In oneaspect, a composition comprising a TFPI-inhibitory peptide isadministered intravenously, intraarterially, or intraperitoneally tointroduce the peptide of the invention into circulation. Non-intravenousadministration also is appropriate, particularly with respect to lowmolecular weight therapeutics. In certain circumstances, it is desirableto deliver a pharmaceutical composition comprising the TFPI-inhibitorypeptide orally, topically, sublingually, vaginally, rectally, pulmonary;through injection by intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraportal,intralesional, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intranasal, urethral, or enteral means; bysustained release systems; or by implantation devices. If desired, theTFPI-inhibitory peptide is administered regionally via intraarterial orintravenous administration feeding a region of interest, e.g., via thefemoral artery for delivery to the leg. In one embodiment, the peptideis incorporated into a microparticle as described in, for example, U.S.Pat. Nos. 5,439,686 and 5,498,421, and U.S. Patent Publications2003/0059474, 2003/0064033, 2004/0043077, 2005/0048127, 2005/0170005,2005/0142205, 2005/142201, 2005/0233945, 2005/0147689. 2005/0142206,2006/0024379, 2006/0260777, 2007/0207210, 2007/0092452, 2007/0281031,and 2008/0026068. Alternatively, the composition is administered viaimplantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Wherean implantation device is used, the device in one aspect is implantedinto any suitable tissue, and delivery of the desired molecule is invarious aspects via diffusion, timed-release bolus, or continuousadministration. In other aspects, the TFPI-inhibitory peptide isadministered directly to exposed tissue during surgical procedures ortreatment of injury, or is administered via transfusion of bloodprocedures. Therapeutic delivery approaches are well known to theskilled artisan, some of which are further described, for example, inU.S. Pat. No. 5,399,363.

To facilitate administration, the TFPI-binding peptide (e.g.,TFPI-inhibitory peptide) in one embodiment is formulated into aphysiologically-acceptable composition comprising a carrier (i.e.,vehicle, adjuvant, buffer, or diluent). The particular carrier employedis limited only by chemico-physical considerations, such as solubilityand lack of reactivity with the peptide, and by the route ofadministration. Physiologically-acceptable carriers are well known inthe art. Illustrative pharmaceutical forms suitable for injectable useinclude without limitation sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions (for example, see U.S. Pat. No. 5,466,468).Injectable formulations are further described in, e.g., Pharmaceuticsand Pharmacy Practice, J. B. Lippincott Co., Philadelphia. Pa., Bankerand Chalmers. eds., pages 238-250 (1982), and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Apharmaceutical composition comprising a peptide provided herein isoptionally placed within containers, along with packaging material thatprovides instructions regarding the use of such pharmaceuticalcompositions. Generally, such instructions include a tangible expressiondescribing the reagent concentration, as well as, in certainembodiments, relative amounts of excipient ingredients or diluents thatmay be necessary to reconstitute the pharmaceutical composition.

When appropriate, the TFPI-binding peptide (e.g., TFPI-inhibitorypeptide) of the invention is administered in combination with othersubstances and/or other therapeutic modalities to achieve an additionalor augmented biological effect. Co-treatments include, but are notlimited to, plasma-derived or recombinant coagulation factors,hemophilia prophylaxis treatments, immunosuppressants, plasmafactor-inhibiting antibody antagonists (i.e., anti-inhibitors),antifibrinolytics, antibiotics, hormone therapy, anti-inflammatoryagents (e.g., Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) orsteroidal anti-inflammatory substances), procoagulants, and painrelievers. In one aspect, the method is an adjunct therapy totraditional replacement factor treatment regimens involvingadministration of, e.g., FXIII, FXII, FXI (e.g., HEMOLEVEN® (Laboratoirefrancais du Fractionnement et des Biotechnologies, Les Ulis, France) andFXI concentrate (BioProducts Laboratory, Elstree, Hertfordshire, UK)),FX, FIX (e.g., BENEFIX® Coagulation Factor IX (Wyeth, Madison, N.J.);ALPHANINE® SD (Grifols, Los Angeles, Calif.); MONONINE® (CSL Behring,King of Prussia, Pa.); BEBULIN-VH™ (Baxter, Deerfield, Ill.);PROFILNINE® SD (Grifols, Los Angeles, Calif.); or PROPLEX T™ (Baxter,Deerfield, Ill.)), FVIII (e.g., ADVATE™ (Baxter, Deerfield, Ill.);HELIXATE® FS(CSL Behring, King of Prussia, Pa.); REFACTO® (Wyeth,Madison, N.J.), XYNTHA™ (Wyeth, Madison, N.J.), KOGENATE® and KOGENATE®FS (Bayer, Pittsburgh, Pa.); ALPHANATE® (Grifols, Los Angeles, Calif.);HEMOPHIL M™ (Baxter, Deerfield, Ill.); KOATE®-DVI (TalecrisBiotherapeutics-USA, Research Triangle Park, N.C.); or MONARC-M™(Baxter, Deerfield, Ill.)), FVIIa (e.g., NOVOSEVEN® FVIIa (Novo Nordisk,Princeton, N.J.) and FVII concentrate (Baxter Bioscience, Vienna,Austria, or BioProducts Laboratory, Elstree, Hertfordshire, UK)), FV,FVa, FII, and/or FIII, to a subject. In some instances, the subject alsoreceives FEIBA VH Immuno™ (Baxter BioScience, Vienna, Austria), which isa freeze-dried sterile human plasma fraction with Factor VIII inhibitorbypassing activity. FEIBA VH Immuno™ contains approximately equal unitsof Factor VIII inhibitor bypassing activity and Prothrombin ComplexFactors (Factors II, VII, IX, and X and protein C). Other exemplaryco-treatments include, but are not limited to, prekallikrein, highmolecular weight kininogen (HMWK), Von Willebrand's factor, TissueFactor, and thrombin. Alternatively or in addition, the TFPI-inhibitorypeptide is co-formulated with one or more different TFPI-inhibitorypeptides. In one aspect, administration of the TFPI-binding peptideallows a reduction in the dose of co-therapeutic required to achieve adesired biological response.

The invention thus includes administering to a subject a TFPI-bindingpeptide (e.g., TFPI-inhibitory peptide) of the invention (or multipleTFPI-inhibitory peptides), in combination with one or more additionallysuitable substances(s), each being administered according to a regimensuitable for that medicament. Administration strategies includeconcurrent administration (i.e., substantially simultaneousadministration) and non-concurrent administration (i.e., administrationat different times, in any order, whether overlapping or not) of theTFPI-inhibitory peptide and one or more additionally suitable agents(s).It will be appreciated that different components are optionallyadministered in the same or in separate compositions, and by the same ordifferent routes of administration.

In some embodiments, the peptide of the invention is conjugated to amoiety, e.g., a therapeutic or diagnostic moiety, such as the detectionmoieties and co-treatments described above. Alternatively or inaddition, the peptide is administered in combination with an interactionpartner (e.g., an antibody, antibody fragment, anticalin, aptamer, orspiegelmer) that (a) binds the peptide and (b) is therapeutically activeand/or is linked to a moiety that provides additional functionality tothe interaction partner (e.g., a therapeutic, diagnostic, or detectionagent). Suitable moieties include, but are not limited to,photosensitizers, dyes, radionuclides, radionuclide-containingcomplexes, enzymes, toxins, antibodies, antibody fragments, andcytotoxic agents, and, in some instances, the moiety possessestherapeutic activity (i.e., achieves an advantageous or desiredbiological effect). The peptide conjugates or peptide-interactionpartner pair is suitable for use in any of the methods described herein,such as methods of treating a subject suffering from a disease ordisorder or at risk of suffering from a disease or disorder.

The invention further provides a method for identifying a TFPI-bindingcompound, such as a TFPI-binding peptide. In one aspect, the methodcomprises (a) contacting a peptide comprising TFPI Kunitz domain 1 (KD1)with a TFPI-binding peptide described herein and a test compound underconditions that allow formation of KD1-TFPI-binding peptide complexes.The method further comprises (b) measuring KD1-TFPI-binding peptidecomplexes formed in step (a), and (c) comparing the number ofKD1-TFPI-binding peptide complexes formed in the presence of the testcompound with the number of KD1-TFPI-binding peptide complexes formed inthe absence of the test compound. A reduction in the number ofKD1-TFPI-binding peptide complexes formed in the presence of the testcompound compared to the number of KD1-TFPI-binding peptide complexesformed in the absence of the test compound indicates that the testcompound is a TFPI-binding compound. In one aspect, the method furthercomprises forming KD1-TFPI-binding complexes in the absence of the testcompound for comparison in step (c), although this is not requiredinasmuch as the information may be obtained separately (e.g., frompreviously prepared reference standards).

KD1, the TFPI-binding peptide, and the test compound are combinedsimultaneously or sequentially, optionally with washing steps beforeand/or after addition of the TFPI-binding peptide and/or the testcompound. In one embodiment, the peptide comprising KD1 is contactedwith a TFPI-binding peptide described herein under conditions that allowformation of KD1-TFPI-binding peptide complexes, unbound TFPI-bindingpeptide is removed, and the remaining KD-peptide complexes are contactedwith a test compound. Displacement of the TFPI-binding peptide from theTFPI-peptide complexes is detected, and indicates that the test compoundis a TFPI-binding compound. Displacement is detected by, for example,measuring the number of KD1-TFPI-binding peptide complexes before andafter exposure to the test compound.

KD1-TFPI-binding peptide complexes are detected and/or measured(quantified) using any suitable detection means, including detectionmeans known in the art for detecting peptides in a sample. For example,in one embodiment of the invention, the TFPI-binding peptide comprises alabel that generates a signal. Exemplary labels are described herein andinclude, e.g., radionuclides, fluorescent dyes, isotopes, enzymesubstrates, and enzymes. The method comprises measuring signal generatedby KD1-TFPI-binding peptide complexes and comparing signal generated byKD1-TFPI-binding peptide complexes formed in the presence of the testcompound with signal generated by KD1-TFPI-binding peptide complexesformed in the absence of the test compound. A reduction in signal from asample comprising KD1-TFPI-binding peptide complexes exposed to testcompound (compared to signal generated by a similar sample ofKD1-TFPI-binding peptide complexes not exposed to the test compound)indicates that complex formation has been inhibited or disrupted, andthat the test compound is a TFPI-binding compound.

The invention also provides a method of identifying a TFPI-bindingcompound that interferes with TFPI-FXa interactions. The method ispredicated, at least in part, on the surprising discovery that TFPI KD1binds to an exosite of FXa and contributes to TFPI's inhibition of FXaactivity. In one aspect, the method comprises contacting a peptideconsisting essentially of KD1 (i.e., a peptide comprising KD1 in theabsence of KD2) with FXa in the presence of a test compound underconditions that allow binding of KD1 to FXa. The method furthercomprises comparing KD1-FXa binding in the presence of the test compoundwith KD1-FXa binding in the absence of the test compound. A decrease inKD1-FXa binding in the presence of the test compound compared to KD1-FXabinding in the absence of the test compound indicates that the testcompound is a TFPI-binding compound. KD1-FXa binding can be detectedand/or quantitated using any method, such as the methods describedherein. For example, KD1 or FXa is labeled, and the signal generated byKD1-FXa complexes exposed to the test compound is compared to the signalgenerated by KD1-FXa complexes not exposed to the test compound.

The methods of the invention to identify TFPI-binding compounds areparticularly amenable to the various high throughput screeningtechniques known in the art. Any “test compound” (e.g., small molecule,peptide, protein (such as an antibody or fragment thereof),peptidomimetic, or polynucleotide (DNA or RNA)) is suitable forscreening using the methods described herein. If desired, a collection,population, or library of test compounds is screened for TFPI binding(and, optionally, anti-TFPI activity) using the methods describedherein. There are a number of different libraries used for theidentification of TFPI inhibitors, including, but not limited to,chemical libraries, natural product libraries, and combinatoriallibraries comprising peptides and/or organic molecules. A chemicallibrary, in some aspects, consists of structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” viaother screening methods. Natural product libraries are collections ofsubstances isolated from or produced by microorganisms, animals, plants,or marine organisms. Combinatorial libraries are composed of largenumbers of peptides or organic compounds, typically as a mixture. Themethods described herein also are useful for screening a display ornucleic acid library, such as a yeast display library, a bacterialdisplay library, a phage display library, a ribosome display library, anmRNA display library, a RNA library, or a DNA library. One method ofscreening a display library is exemplified in Example 1. High throughputscreening methods embraced by the invention include automated proceduresallowing screening of tens to hundreds of thousands of test compounds.

In another aspect, the inventive method for identifying a TFPI-bindingcompound comprises contacting a peptide comprising (or consisting of)KD1 with a test compound, and detecting binding of the test compound toa TFPI binding site defined by KD 1 amino acid residues corresponding tohuman TFPI residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55,such as a binding site defined by human TFPI residues Ala27, Phe28,Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55. Inone embodiment, the binding site is defined by amino acid residuescorresponding to human TFPI residues Ala27, Phe28, Lys29, Ala30, Asp31,Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55. The bindingsite corresponds to the TFPI binding site of JBT1857, a TFPI-bindingpeptide that inhibits TFPI activity in a number of functional assays.

The TFPI binding site amino acid residues described herein are inreference to the human TFPI amino acid sequence, and the numberingrefers to the position of the recited amino acid in relation to theN-terminus of human TFPI. Merely for the purpose of illustrating theposition of the TFPI binding site, the amino acid sequence of a fragmentof human TFPI comprising KD1 is provided as SEQ ID NO: 4234(DSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIGGCEGNQNRFESLEECKKMCTRDNA (amino acids 26-75 encoding KD1 are indicated inbold)). Corresponding amino acids of other TFPI polypeptides (such asTFPI polypeptides from different organisms, or TFPI polypeptidefragments) are identified, for example, by aligning a polypeptide'samino acid sequence with SEQ ID NO: 4234. While, in one embodiment, thepeptide comprising TFPI KD1 does not comprise other regions of the TFPIprotein responsible for TFPI activity, other embodiments entail the useof a peptide comprising amino acids 1-160 of human TFPI (comprising KD1and KD2) or comprising full length human TFPI (containing KD1-KD3).

Binding of a test compound to the TFPI binding site defined herein isdetected using any of a number methods, including the detection methodsdescribed herein. An exemplary method for detecting binding employsnuclear magnetic resonance (NMR) to recognize chemical shifts at aminoacid residues within the TFPI binding site. Chemical shifts at TFPIamino acid positions 28-30, 32, 46, 47, and 55, and optionally positions27, 31, 36-38, and 44, denotes interaction of the test compound withthese amino acid contact points on TFPI. To determine the presence orabsence of chemical shifts at particular amino acids resulting from testcompound binding, NMR data obtained from the KD1-test compound complexis compared to NMR data obtained from free KD1 peptide. Use of NMR todetect binding between a test compound and TFPI KD1 is further describedin the Examples.

Alternatively, binding of a test compound to the TFPI-binding sitedefined herein is determined indirectly by detecting alterations in theability of TFPI KD1 to interact with its natural binding partners, e.g.,FVIIa or FXa. In this regard, the method comprises contacting thepeptide comprising TFPI KD1 with FVIIa in the presence of the testcompound under conditions that allow binding of KD1 to FVIIa, andKD1-FVIIa binding is compared with KD1-FVIIa binding in the absence ofthe test compound. Alternatively or in addition, the method comprisescontacting the peptide comprising TFPI KD1 with FXa in the presence ofthe test compound under conditions that allow binding of KD1 to FXa, andcomparing KD1-FXa binding in the presence of the test compound withKD1-FXa binding in the absence of the test compound. Optionally, thepeptide comprising KD1 also comprises KD2, and the method comprisescontacting the peptide with FXa in the presence of a test compound underconditions that allow binding of KD2 to FXa, and KD2-FXa binding iscompared with KD2-FXa binding in the absence of the test compound. Adecrease in KD1-FVIIa binding, KD1-FXa binding, or KD2-FXa binding inthe presence of the test compound (compared to KD1-FVIIa binding,KD1-FXa binding, or KD2-FXa binding in the absence of the test compound)indicates that the test compound is a TFPI-binding compound. The methodoptionally comprises contacting KD1 and/or KD2 to FVIIa and/or FXa inthe absence of the test compound as a reference for comparing binding inthe presence of the test compound.

KD binding to FVIIa or FXa is determined and/or quantified using anysuitable method for detecting protein-protein interactions, such as themethods described herein using detectable labels. Binding of the testcompound to the TFPI binding site is, alternatively, detected using anenzymatic assay. FVIIa or FXa enzymatic activity is a suitable surrogatefor evaluating binding of the proteins to TFPI KD1 or KD2; testcompounds that bind the TFPI-binding site defined herein inhibit TFPIactivity, resulting in increased FVIIa and FXa activity. Enzymaticassays for evaluating FVIIa or FXa activity are described in detailherein.

The invention further includes compounds identified as TFPI-bindingcompounds in the methods of the invention, as well as compositionscomprising one or more identified compounds. Methods for isolating orpurifying a compound, such as TFPI-binding compound (e.g., aTFPI-binding peptide) identified as described herein are known in theart and described above. In some aspects, TFPI-binding compoundsidentified as described herein are TFPI inhibitors that downregulate orablate one or more TFPI activities. In one embodiment, the inventionincludes a method for purifying a compound that inhibits FXa activity.The method comprises contacting a peptide comprising TFPI KD1 with acompound under conditions that allow formation of compound-KD1complexes, removing unbound compound, and dissociating the compound-KD1complexes to release the compound, which binds TFPI. Use of a TFPIinhibitor identified and/or purified as described herein for themanufacture of a medicament, such as a medicament for treating a bloodcoagulation disorder, is provided, as well as a method for treating asubject suffering from a disease or at risk of suffering from a diseasecomprising administering the TFPI inhibitor to the subject.

In addition, a method of inhibiting human TFPI is provided, wherein themethod comprises contacting human TFPI with an inhibitor that bindshuman TFPI at a binding site defined by amino acid residues Phe28,Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55. Another aspect of theinvention includes a method for treating a subject suffering from adisease or at risk of suffering from a disease. The method comprisesadministering to the subject an inhibitor that binds human TFPI at abinding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32,Ile46, Phe47, and Ile55. In one aspect, the human TFPI binding site isdefined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32,Lys36, Ile38, Ile46, Phe47, and Ile55, such as a binding site defined byamino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36,Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55. Any inhibitor thatcontacts the TFPI binding site defined herein and inhibits(downregulates or ablates) one or more TFPI activity is suitable for usein the context of the method. The TFPI inhibitor is, optionally, aTFPI-binding peptide, such as a TFPI-binding peptide having thecharacteristics described herein.

The invention further includes computer storage media and methods formodeling candidate TFPI-compounds in the TFPI binding site definedherein. Three dimensional (3D) modeling of proteins can be used inconjunction with 3D models of various test TFPI-binding compounds (e.g.,peptides or small molecules) to determine fit between the compounds andtargeted amino acids in TFPI. Because the effectiveness of a testcompound in inhibiting TFPI can be limited if the compound does notremain attached to TFPI for a sufficient period of time to effect abiological response, the tendency of the two to remain coupled can bepredicted to develop an affinity rating.

By analyzing the 3D surface of the TFPI protein and the fit of thecorresponding compound to the surface in view of the affinity rating,modifications to the compound (e.g., peptide) can be developed toimprove both the number of contact points between the surface and thecompound and the strength of the bonds at the contact points. Theeffectiveness of chemical-based candidates and peptide-based TFPIinhibitors can similarly be modeled using this technique, whichfacilitates the rational design of TFPI-binding compounds. A computermodel of the three dimensional (3D) surface of KD1 allows testing of theability of various peptides or chemicals to attach to an identifiedsubset of amino acids that define a TFPI binding site and inhibit KD 1.A surface of the KD 1 protein is modeled in 3D space on a computer,particularly a surface bounded by the targeted amino acids in KD 1. The3D models of various peptides, for example, can be matched to thesurface to determine how many of the target TFPI amino acids arecontacted by the peptide and also to develop an affinity ratingpredicting how long the peptide will remain attached to the targetsurface.

By changing the peptide model and repeating the computer modeling,affinity ratings can be quickly generated for a peptide family. The mostpromising peptide variants (e.g., a second peptide comprising one ormore substitutions within the amino acid sequence of a parent peptide)can be singled out for further physical testing, if desired.

The invention provides a computer storage media having computerexecutable instructions that, when executed on the processor of acomputer, implement a method of modeling interaction between selectedthree dimensional (3D) points in a TFPI KD 1 protein and a testcompound. The method comprises obtaining a protein structure 3D modelfor the TFPI KD1 protein; determining a 3D relationship between aselected subset of amino acids in the protein structure, wherein theselected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32,Ile46, Phe47, and Ile55; modeling a surface bounded by the selectedsubset of amino acids; obtaining a test compound 3D model of a testcompound; matching the test compound 3D model to the surface bounded bythe selected subset of amino acids; and identifying contact pointsbetween the selected subset of amino acids of the surface and the testcompound 3D model. Optionally, the method further comprises determininga number of the contact points between the surface and the test compound3D model; and recording an affinity rating for the test compound 3Dmodel corresponding to the number of contact points. In one aspect, theselected subset of amino acids comprises (or consists of) Ala27, Phe28,Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47,and Ile55. The method further optionally comprises obtaining an updatedtest compound 3D model based on a second test compound; matching theupdated test compound 3D model to the surface bounded by the selectedsubset of amino acids; and identifying the identified contact pointsbetween the selected subset of amino acids of the surface and theupdated test compound 3D model on a display of the computer. In oneembodiment, the method further comprises determining a number of thecontact points between the surface and the updated test compound 3Dmodel; determining a bond type for each contact point between thesurface and the updated test compound 3D model; and recording a newaffinity rating based on the number of contact points and an aggregateof the bond types for each contact point between the surface and theupdated test compound 3D model. The updated affinity rating is thencompared with the new affinity rating to determine whether the testcompound or the second test compound has a higher affinity rating, ifdesired. The contact points can be displayed on the computer, therebyfacilitating optimization or design of TFPI-binding compounds.

In another embodiment, the computer storage media has computerexecutable instructions that, when executed on the processor of acomputer, implement a method of comparing a peptide to selected threedimensional points (3D) in a TFPI Kunitz domain 1 protein (KD 1), themethod comprising creating a protein structure for the KD 1 protein;determining a three dimensional model of a selected subset of aminoacids in the KD1 protein, wherein the subset of amino acids comprisesPhe28, Lys29, Ala30, Asp32, Ile46, Phe47 and Ile55; determining a threedimensional model of a peptide; fitting the 3D model of the peptide tothe 3D model of the selected subset of amino acids; and generating anaffinity of the peptide for the selected subset of amino acids, whereinthe affinity is based on a number of amino acids in the subset incontact with the peptide and a bond strength at each contact point.

In addition, a method of comparing a test compound to selected threedimensional points in a TFPI KD1 protein is provided. The methodcomprises creating a protein structure for the KD 1 protein in a memoryof a computer; determining a three dimensional model of a selectedsubset of amino acids in the KD1 protein at a processor of the computer,wherein the selected subset of amino acids comprises Phe28, Lys29,Ala30, Asp32, Ile46, Phe47, and Ile55; determining a three dimensionalmodel of a test compound at the processor of the computer; fitting the3D model of the test compound to the 3D model of the selected subset ofamino acids at the processor of the computer; and generating an affinityof the test compound for the selected subset of amino acids at theprocessor of the computer, wherein the affinity is based on a number ofamino acids in the subset in contact with the test compound and a bondstrength at each contact point. The method further comprises, in someembodiments, displaying a 3D representation of the fit between the testcompound and the 3D model of the selected subset of amino acids and,optionally, repeating the steps described herein for a plurality of testcompounds and saving the respective affinities for each of the pluralityof test compounds.

With reference to FIG. 58, an exemplary system for implementing theclaimed method and apparatus includes a general purpose computing devicein the form of a computer 110. Components shown in dashed outline arenot technically part of the computer 110, but are used to illustrate theexemplary embodiment of FIG. 58. Components of computer 110 may include,but are not limited to, a processor 120, a system memory 130, amemory/graphics interface 121 and an I/O interface 122. The systemmemory 130 and a graphics processor 190 may be coupled to thememory/graphics interface 121. A monitor 191 or other graphic outputdevice may be coupled to the graphics processor 190.

A series of system busses may couple various system components includinga high speed system bus 123 between the processor 120, thememory/graphics interface 121 and the I/O interface 122, a front-sidebus 124 between the memory/graphics interface 121 and the system memory130, and an advanced graphics processing (AGP) bus 125 between thememory/graphics interface 121 and the graphics processor 190. The systembus 123 may be any of several types of bus structures including, by wayof example, and not limitation, such architectures include IndustryStandard Architecture (USA) bus, Micro Channel Architecture (MCA) busand Enhanced ISA (EISA) bus. As system architectures evolve, other busarchitectures and chip sets may be used but often generally follow thispattern. For example, companies such as Intel and AMD support the IntelHub Architecture (IHA) and the Hypertransport™ architecture,respectively.

The computer 110 typically includes a variety of computer readablemedia. Computer readable media can be any available media that can beaccessed by computer 110 and includes both volatile and nonvolatilemedia, removable and non-removable media. By way of example, and notlimitation, computer readable media may comprise computer storage media.Computer storage media includes both volatile and nonvolatile, removableand non-removable media implemented in any method or technology forstorage of information such as computer executable instructions, datastructures, program modules or other data. Computer storage mediaincludes RAM, ROM, EEPROM, flash memory or other memory technology,CD-ROM, digital versatile disks (DVD) or other optical disk storage,magnetic cassettes, magnetic tape, magnetic disk storage or othermagnetic storage devices or other physical storage elements thatphysically embody electronic data and excludes any propagated media suchas radio waves or modulated carrier signals.

The system memory 130 includes computer storage media in the form ofvolatile and/or nonvolatile memory such as read only memory (ROM) 131and random access memory (RAM) 132. The system ROM 131 may containpermanent system data 143, such as computer-specific configuration data.RAM 132 typically contains data and/or program modules that areimmediately accessible to and/or presently being operated on byprocessor 120. By way of example, and not limitation, FIG. 58illustrates operating system 134, application programs 135, otherprogram modules 136, and program data 137.

The I/O interface 122 may couple the system bus 123 with a number ofother busses 126, 127 and 128 that couple a variety of internal andexternal devices to the computer 110. A serial peripheral interface(SPI) bus 126 may connect to a basic input/output system (BIOS) memory133 containing the basic routines that help to transfer informationbetween elements within computer 110, such as during start-up.

A super input/output chip 160 may be used to connect to a number of‘legacy’ peripherals, such as floppy disk 152, keyboard/mouse 162, andprinter 196, as examples. The super I/O chip 160 may be connected to theI/O interface 122 with a bus 127, such as a low pin count (LPC) bus, insome embodiments. Various embodiments of the super I/O chip 160 arewidely available in the commercial marketplace. In one embodiment, bus128 may be a Peripheral Component Interconnect (PCI) bus.

The computer 110 may also include other removable/non-removable,volatile/nonvolatile computer storage media. By way of example only,FIG. 58 illustrates a hard disk drive 140 that reads from or writes tonon-removable, nonvolatile magnetic media. The hard disk drive 140 maybe a conventional hard disk drive.

Removable media, such as a universal serial bus (USB) memory 153,firewire (IEEE 1394), or CD/DVD drive 156 may be connected to the PCIbus 128 directly or through an interface 150. Otherremovable/non-removable, volatile/nonvolatile computer storage mediathat can be used in the exemplary operating environment include, but arenot limited to, magnetic tape cassettes, flash memory cards, digitalversatile disks, digital video tape, solid state RAM, solid state ROM,and the like.

The drives and their associated computer storage media discussed aboveand illustrated in FIG. 58, provide storage of computer readableinstructions, data structures, program modules and other data for thecomputer 110. In FIG. 58, for example, hard disk drive 140 isillustrated as storing operating system 144, application programs 145,other program modules 146, and program data 147. Note that thesecomponents can either be the same as or different from operating system134, application programs 135, other program modules 136, and programdata 137. Operating system 144, application programs 145, other programmodules 146, and program data 147 are given different numbers here toillustrate that, at a minimum, they are different copies. A user mayenter commands and information into the computer 20 through inputdevices such as a mouse/keyboard 162 or other input device combination.Other input devices (not shown) may include a microphone, joystick, gamepad, satellite dish, scanner, or the like. These and other input devicesare often connected to the processor 120 through one of the I/Ointerface busses, such as the SPI 126, the LPC 127, or the PCI-128, butother busses may be used. In some embodiments, other devices may becoupled to parallel ports, infrared interfaces, game ports, and the like(not depicted), via the super I/O chip 160.

The computer 110 may operate in a networked environment using logicalcommunication ports to one or more remote computers, such as a remotecomputer 180 via a network interface controller (NIC) 170. The remotecomputer 180 may be a personal computer, a server, a router, a networkPC, a peer device or other common network node, and typically includesmany or all of the elements described above relative to the computer110. The logical connection between the NIC 170 and the remote computer180 depicted in FIG. 58 may include a local area network (LAN), a widearea network (WAN), or both, but may also include other networks. Suchnetworking environments are commonplace in offices, enterprise-widecomputer networks, intranets, and the Internet.

FIG. 59 illustrates a 3D model of a TFPI protein 200 showingrepresentative amino acids 202, 204, 206 that comprise the TFPI protein.A specific region of the TFPI protein of interest is KD1, notspecifically illustrated. The surface shown is formed by the placementof the amino acids making up the protein. The surface of formed byspecific amino acids in the KD 1 region are of interest when studying orcreating a TFPI inhibitor. As discussed in more detail herein, thebiological effects of KD1 are inhibited by binding certain amino acidsof within the KD1 region. Specifically, these target amino acids includeAla27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44,Ile46, Phe47, and Ile55.

FIG. 60 illustrates a peptide 300 that binds to at least a portion ofthe target amino acids listed above.

FIG. 61 is an illustration of a method of performing KD 1 and peptideinteraction modeling.

A 3D model of a protein may be obtained (block 302) and stored on amemory 140 of a computer 110. The model may be generated locally using aknown tool or may be obtained from a public source. In one embodimentthe protein is TFPI KD1 200.

A 3D relationship between a selected subset of amino acids in theprotein structure may be determined (block 304). In one embodiment, theselected subset of amino acids comprises Phe28, Lys29, Ala30, Asp32,Ile46, Phe47 and Ile55; and optionally further comprises Ala27, Asp31,Lys36, and Ile38; and optionally further comprises Ala37 and Phe44,although not every amino acid listed here is required for binding tohave an inhibitory (e.g., therapeutic) effect. That is, further subsetsof this group may also have properties of interest.

For the particular subset of amino acids of interest, a surface boundedby the selected subset of amino acids may be modeled. An outer perimetermay be defined by those amino acids not having further amino acids ofinterest on each side. A texture of the surface may be defined by the 3Dlocation of each amino acid in the subset (block 306).

A 3D model of a candidate TFPI-binding compound (e.g., peptide) ofinterest may be generated and stored at a memory 140 of the computer 110(block 308).

The peptide 3D model may be matched or fitted to the surface bounded bythe selected subset of amino acids (block 310). A best fit between thetwo may be developed at the points of interest, that is, on the selectedamino acids of KD1. Several computer tools are available for such 3Dmodeling and fitting and may be used to create 3D models and match oneto another. One example is the HADDOCK tool described in: “de Vries, S.J., van Dijk, A. D. J., Krzeminski, M., van Dijk, M., Thureau, A., Hsu,V., Wassenaar, T. and Bonvin, A. M. J. J. (2007), HADDOCK versusHADDOCK: New features and performance of HADDOCK2.0 on the CAPR1targets. Proteins: Structure, Function, and Bioinformatics, 69: 726-733.doi: 10.1002/prot.21723”

The contact points between the model of the surface of the selectedsubset of amino acids of the surface and the test compound (e.g.,peptide) 3D model may be identified, stored, and optionally displayed ona monitor 191 of the computer 110 (block 312). A compound (e.g.,peptide) may be modified to increase the number of contact points or thestrength of the bonds at the contact points. To facilitate modeling thiseffect, a metric, described further below, may be developed to measurethe affinity of the compound to bind to the protein of interest, in ourexample, KD1.

Further, the contact points between the surface and the compound 3Dmodel may be counted (block 314) and an affinity rating for the compound3D model may be recorded corresponding to the number of contact points(block 316). For example, if all 14 of the above listed amino acids aretargeted and 12 of the 14 are actually contacted, or bound, by thecompound 3D model, an affinity rating of 12/14 or 0.86 may be calculatedand recorded.

However, the affinity rating as a measure of how tightly a candidatecompound is coupled, and therefore, how long it may stay coupled to KD1may be more accurately described in terms of not only the number ofbonds of interest but also the type of bond. The bond type for eachcontact point may also be determined (block 318). With respect toTFPI-binding peptides, hydrophobic bonds having an intermoleculardistant of ≦4 angstroms may be differentiated from bonds with anintermolecular distance of 2.6-3.2 angstroms. In one embodiment, bondsless than 3.2 angstroms may be assigned a weight of 1.5 and bonds>than3.2 angstroms may be assigned a weight of 1.25. The affinity rating maybe updated or recalculated in view of the bond type using this, oranother weighting (block 320). For example, if, in the previous example,5 of the bonds are short bonds and 7 of the bonds are long bonds, thenew affinity rating may be (5*1.5+7*1.25)/14=1.16.

If only 7 amino acids from KD 1 are targeted and 4 connect with shortbonds, the affinity rating may be (4*1.5)/7=0.86. However, in this casethe fewer targeted amino acids will be considered when comparisons aremade to other affinity ratings. For example, all ratings could benormalized to a standard based on total desired target sites.

If no more iterations are to be performed the no branch from block 322may be taken and the results of may be stored for future analysis anddecision making (block 324). If additional peptides, or variants of thepreviously tested peptide, are to be analyzed, the yes branch from block322 may be taken and a new or updated model of the peptide of interestmay be generated or otherwise obtained and stored (block 326). The stepsat blocks 310 to 320 may be repeated and the results of the current runmay be compared to results from previous runs to determine whichpeptides/variants have higher affinity ratings and merit more work,including possible physical testing.

The ability to target particular sites with 3D modeling and to generatea comparative rating allows hundreds, if not thousands of samples to beprocessed and compared with relative ease, avoiding the time and cost ofx-ray crystallography. This technique may be particularly applicable tomodeling associated with the Phe28, Lys29, Ala30, Asp32, Ile46, Phe47,Ile55, Ala27, Asp31, Lys36, Ile38, Phe2, Ala37 and Phe44 amino acidsfrom TFPI KD 1.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. In addition, the entiredocument is intended to be related as a unified disclosure, and itshould be understood that all combinations of features described hereinare contemplated, even if the combination of features are not foundtogether in the same sentence, or paragraph, or section of thisdocument. For example, where protein therapy is described, embodimentsinvolving polynucleotide therapy (using polynucleotides/vectors thatencode the protein) are specifically contemplated, and the reverse alsois true. Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The invention includes, for instance,all embodiments of the invention narrower in scope in any way than thevariations specifically mentioned above. With respect to aspects of theinvention described as a genus, all individual species are individuallyconsidered separate aspects of the invention. With respect to aspects ofthe invention described or claimed with “a” or “an,” it should beunderstood that these terms mean “one or more” unless contextunambiguously requires a more restricted meaning. With respect toelements described as one or more within a set, it should be understoodthat all combinations within the set are contemplated. Finally, unless aclaim element is defined by reciting the word “means” and a functionwithout the recital of any structure, it is not intended that the scopeof any claim element be interpreted based on the application of 35U.S.C. §112, sixth paragraph.

EXAMPLES

The invention, thus generally described, will be understood more readilyby reference to the following examples, which are provided by way ofillustration and are not intended to limit the invention.

Example 1

The following example describes production, identification, andscreening of peptides for binding to TFPI.

Peptides candidates were obtained from commercial suppliers (e.g.,PolyPeptide Laboratories SAS (Strasbourg, France) and JPT PeptideTechnologies GmbH (Berlin, Germany)). Methods for synthesizing candidatepeptides are provided above. Candidate peptides were synthesized astrifluoroacetate (TFA) salts with a purity>90% or >60%. All peptideswere solved in DMSO to a stock concentration of 10 mM. TFPI-bindingpeptide sequences were identified using an mRNA display library. ThemRNA display technology is superior to other library screeningtechniques for allowing for a diversity of 10¹⁴ different sequenceswithin a starting pool and avoiding, e.g., the in vivo steps requiredfor phage display. In brief, the technology involves directly linkingmRNA to its encoded candidate peptide through a puromycin molecule (FIG.5). The mRNA display method is further described in International PatentPublication No. WO 2005/051985 and Liu et al., Methods in Enzymology,318, 268-293 (2000). TFPI was immobilized to a solid support via biotinand exposed to candidate peptide-RNA complexes. TFPI-bound candidatepeptide-RNA complexes were isolated, and the RNA reverse transcribed toobtain coding DNA. High affinity binders were obtained following six toten selection rounds using a competitive elusion strategy. Many of thecandidate peptides were 31 amino acids in length (27 randomized aminoacids and 2 amino acids flanking both termini).

Selected peptides were synthesized and subjected to peptide optimizationusing a microarray-based scan analysis to identify peptide fragmentsretaining TFPI-binding affinity. For example, a microarray-based scan ofJBT0047 was performed using a series of 20 amino acid fragments of thepeptide, the sequences of which overlapped by 19 amino acids. Briefly,N-terminally, aminooxyacetate-modified peptides were printed on Corningepoxide glass slides. After washing and drying, the slides were treatedin a TECAN HS400™ incubation station. Slides were washed for two minutesin Tris-buffered saline with 0.1% TWEEN 20® (TBST), and blocked for 30minutes in Tris-based, T-20 SuperBlock™ buffer (5 mM CaCl₂) (Pierce).After blocking, the slides were washed for 2.5 minutes in TBST. Theslides were subsequently incubated with DYLIGHT™ 649-labeled TFPI (1μg/ml in Tris-based, T-20 SuperBlock™ buffer (5 mM CaCl₂)) for 45minutes, and washed twice with continuous flow TBST for ten minutes. Theslides were subjected to a final wash with saline-sodium citrate bufferfor two minutes, and air-dried for four minutes. The slides were scannedin an Axon GenePix® 4000B scanner, and scans were analyzed using theGenePix® Pro software. N- and C-terminal truncation analysissupplemented the scan analysis. The microarray scan results demonstratedthat peptide JBT0293 bound TFPI with the highest affinity. A series ofsubstitution mutants based on the amino acid sequence of JBT0293 wasgenerated and tested for TFPI binding properties.

The affinity of a subset of peptides for TFPI was demonstrated via anenzyme-linked immunosorbent assay (ELISA)-like assay (binding (EC₅₀)ELISA) performed with biotinylated peptides. Ninety-six well MaxiSorpplates (Nunc) were coated with 3 μg/mL TFPI in coating buffer (15 mMNa₂CO₃, 35 mM NaHCO₃, pH 9.6) over night. Plates were washed three timeswith 350 μl wash buffer (HNaT: 175 mM NaCl, 25 mM HEPES, 5 mM CaCl₂,0.1% Tween 80, pH 7.35), and subsequently blocked with 200 μl 2% yeastextract in HNaT for 2 hours. Plates were then washed three times with350 μl HNaT. Biotinylated candidate peptides were diluted from a DMSOstock 1/200 in HNaT. The initial peptide concentration was 50 μM if noprecipitate appeared during the 1/200 dilution of the 10 mM peptidestock solution. Pre-dilutions of the peptide stock in DMSO wereconducted if precipitates formed. The diluted peptides were applied tothe Maxisorp plates, serial dilutions (1/3) were generated, and thedilutions were incubated for 1.5 hours at room temperature. Incubationwas followed by three wash steps (350 μl HNaT). Bound peptide wasdetected by incubation with horseradish peroxidase-conjugatedstreptavidin (1 hour), followed by three wash steps with HNaT and asubsequent chromogenic conversion of added TMB(3,3′5,5′-Tetramethylbenzidin). The assay is illustrated in FIG. 6A.

Generally, peptide binding to immobilized TFPI was significantly abovebackground. EC₅₀ values for biotinylated peptides are given in FIGS.32A-32AM, 33, 34A-34J, 35, 36A-36Q, 37, 38A-38B, and 39. The bindingcurve of one TFPI-binding peptide, JBT0132, is depicted in FIG. 7. TheEC₅₀ of JBT0132 was calculated to be about 2.2 nM.

In addition, a competition (IC₅₀) ELISA was performed using biotinylatedTFPI-binding peptides as “tracers” to compete for TFPI-binding withnon-biotinylated candidate peptides. The assay principle is depicted inFIG. 6B. Ninety-six well MaxiSorp plates (Nunc) were coated with 3 μg/mLTFPI in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) over night.The concentration of TFPI can be altered depending on the particularconditions of the assay; in other IC₅₀ ELISA assays referenced herein,the coating buffer contained 0.05 μg/ml TFPI. Plates were washed threetimes with 350 μl wash buffer (HNaT: 175 mM NaCl, 25 mM HEPES, 5 mMCaCl₂, 0.1% Tween 80, pH 7.35), and blocked with 200 μl 2% yeast extractin HNaT for 2 hours. Plates were then washed three times with 350 μlHNaT. Biotinylated tracer peptides were applied at a concentrationcorresponding to their respective EC₉₀ values determined in the bindingELISA (median if n>2). A competitor stock solution of peptide (10 mM)was diluted 1/33.3 in HNaT without HSA, and a serial 1/3 dilution wasprepared with HNaT with 3% DMSO. The dilution strategy employed in aparticular assay will depend on the affinity of the peptides. Thedilution was further diluted with the biotinylated tracer peptide in aratio of 1:6 (20 μl competitor dilution and 100 μl tracer peptide). Themixture of competitor and tracer peptide was applied to the TFPI-coatedmicrotiter plate and incubated for 1.5 hours. The plates were washedthree times with 350 μl HNaT. Peptide-TFPI binding was detected byapplying HRP-conjugated streptavidin to the microtiter plate, incubatingthe mixture for one hour, washing the plate three times with 350 μlHNaT, applying TMB (3,3′5,5′-Tetramethylbenzidin), and detecting thesubsequent chromogenic conversion of TMB by HRP. IC₅₀ graphs forrepresentative non-biotinylated peptides are provided in FIGS. 8A-8D.IC₅₀ measurements of peptides JBT0303, JBT0120, and JBT0224 are setforth in Table 3.

TABLE 3 Tracer Tracer Concentration Peptide IC₅₀ [μM] n SD Peptide [μM]JBT0303 0.119 2 0.064 JBT0131 0.0409 JBT0120 0.0189 3 0.0044 JBT01240.0718 JBT0224 n.a. 1 JBT0126 0.240

In addition to the competition ELISA (IC₅₀) assay, a screening assay wasemployed to measure higher numbers of peptides in parallel. Thescreening ELISA is similar to the competition IC₅₀ ELISA with theexception that only three different concentrations of the competitorwere employed (300 nM, 100 nM and 33.3 nM for the JBT0047 class, and50000 nM, 16667 nM and 5556 nM for the JBT0122 class). In someinstances, screening results were expressed as percent inhibition of thetracer signal in relation to a competitive peptide (competitive peptideJBT0477 for the JBT0047 family, and competitive peptide JBT1697 for theJBT0122 family). The competition IC₅₀ assay results and the screeningassay results of peptides prepared and screened in accordance with themethods set forth herein are provided in FIGS. 32A-32AM, 33, 34A-34J,35, 36A-36Q, 37, 38A-38B, and 39. The mean IC₅₀ values presented inFIGS. 32A-32AM, 33, 34A-34J, 35, 36A-36Q, 37, 38A-38B, and 39 are basedon a greater number of assays than the values presented in Table 3 and,therefore, the values may differ slightly. The results of the screeningELISA are presented as percent inhibition of tracer peptide JBT0131binding. Several peptides that were analyzed using the IC₅₀ ELISA areclassified in FIGS. 32A-32AM, 33, 34A-34J, 35, 36A-36Q, 37, 38A-38B, and39 according to their binding affinity as set forth in Table 4.

TABLE 4 TFPI competition ELISA IC₅₀ [nM] Group <50 nM A 50 ≦ x < 100 nMB 100 ≦ x < 250 nM C 250 ≦ x < 1000 nM D 1000 ≦ x < 5000 nM E 5000 ≦ x <10000 nM F 10000 ≦ x < 50000 nM G

Exemplary TFPI-binding peptides identified using the methods describedherein are presented in Table 5. Some peptides were biotinylated, andmany comprise N- and C-terminal lysines to promote solubility. Severalpeptides exhibited TFPI-inhibitory activity in model and/or plasmaticassay systems, as described below.

TABLE 5 Peptide Parent Sequence SEQ ID JBT0047 QSKKNVFVFGYFERLRAK 1JBT0047 JBT0047 Ac-SGVGRLQVAFQSKKNVFVFGYFERLRAKLTS-NH2 253 JBT0051JBT0047 Biotinyl-Ttds-SGVGRLQVAFQSKKNVFVFGYFERLRAKLTS-NH2 962 JBT0055JBT0047 Ac-SGVGRLQVAFQSKKNVFVFGYFERLRAKLTS-Ttds-Lys(Biotinyl)- 963 NH2JBT0131 JBT0047 Biotinyl-Ttds-AFQSKKNVFVFGYFLRLRAK-NH2 964 JBT0132JBT0047 Biotinyl-Ttds-FQSKKNVFVFGYFLRLRAKL-NH2 965 JBT0133 JBT0047Biotinyl-Ttds-QSKKNVFVFGYFERLRAKLT-NH2 966 JBT0155 JBT0047Ac-KKSGVGRLQVAFQSKKNVFVFGYFERLRAKLTSKK-NH2 8 JBT0158 JBT0047Ac-KKSGVGRLQVAFQSKKNVFVFGYFLRLRAKKK-NH2 9 JBT0162 JBT0047Ac-KKGRLQVAFQSKKNVFVFGYFERLRAKLTSKK-NH2 10 JBT0163 JBT0047Ac-KKQVAFQSKKNVFVFGYFERLRAKLTSKK-NH2 11 JBT0164 JBT0047Ac-KKFQSKKNVFVFGYFERLRAKLTSKK-NH2 12 JBT0166 JBT0047Biotinyl-Ttds-KKFQSKKNVFVFGYFLRLRAKLKK-NH2 968 JBT0169 JBT0047Ac-KKAFQSKKNVFVFGYFERLRAKKK-NH2 254 JBT0170 JBT0047Ac-KKFQSKKNVFVFGYFLRLRAKLKK-NH2 13 JBT0171 JBT0047Ac-KKQSKKNVFVFGYFLRLRAKLTKK-NH2 255 JBT0174 JBT0047Ac-KKAFQSKKNVFVFGYFERLRAKLKK-NH2 14 JBT0175 JBT0047Ac-KKAFQSKKNVFVFGYFERLRAKLTKK-NH2 182 JBT0293 JBT0047Ac-FQSKKNVFVFGYFLRLRAKL-NH2 256 X₃X₄X₅KX₇NVFX₁₁X₁₂GYX₁₅X₁₆RLRAKX₂₂ 2JBT0294 JBT0047 Ac-YQSKKNVFVFGYFLRLRAKL-NH2 257 JBT0295 JBT0047Ac-FSSKKNVFVFGYFERLRAKL-NH2 713 JBT0296 JBT0047Ac-FQNKKNVFVFGYFLRLRAKL-NH2 407 JBT0297 JBT0047Ac-FQSKNNVFVFGYFLRLRAKL-NH2 183 JBT0298 JBT0047Ac-FQSKQNVFVFGYFLRLRAKL-NH2 747 JBT0299 JBT0047Ac-FQSKKNVFAFGYFLRLRAKL-NH2 408 JBT0300 JBT0047Ac-FQSKKNVFSFGYFERLRAKL-NH2 409 JBT0301 JBT0047Ac-FQSKKNVFTFGYFLRLRAKL-NH2 470 JBT0302 JBT0047Ac-FQSKKNVFVAGYFLRLRAKL-NH2 258 JBT0303 JBT0047Ac-FQSKKNVFVDGYFLRLRAKL-NH2 184 JBT0304 JBT0047Ac-FQSKKNVFVLGYFLRLRAKL-NH2 259 JBT0305 JBT0047Ac-FQSKKNVFVQGYFLRLRAKL-NH2 260 JBT0306 JBT0047Ac-FQSKKNVFVSGYFERLRAKL-NH2 185 JBT0307 JBT0047Ac-FQSKKNVFVYGYFLRLRAKL-NH2 261 JBT0308 JBT0047Ac-FQSKKNVFVFGYKERLRAKL-NH2 411 JBT0309 JBT0047Ac-FQSKKNVFVFGYYERLRAKL-NH2 412 JBT0310 JBT0047Ac-FQSKKNVFVFGYFDRLRAKL-NH2 262 JBT0311 JBT0047Ac-FQSKKNVFVFGYFLRLRAKN-NH2 748 TFVDERLLYFLTIGNMGMYAAQLKF 3 JBT0049JBT0049 Ac-SGNTFVDERLLYFLTIGNMGMYAAQLKFRTS-NH2 3025 JBT0053 JBT0049Biotinyl-Ttds-SGNTFVDERLLYFLTIGNMGMYAAQLKFRTS-NH2 3006 JBT0057 JBT0049Ac-SGNTFVDERLLYFLTIGNMGMYAAQLKFRTS-Ttds-Lysin(biotin)- 3018 NH2 JBT0190JBT0049 Ac-KKSGNTFVDERLLYFLTIGNMGMYAAQLKFRTSKK-NH2 3031 JBT0193 JBT0049Ac-KKSGNTFVDERLLYFLTIGNMGMYAAQLKFKK-NH2 3073 JBT0197 JBT0049Ac-KKTFVDERLLYFLTIGNMGMYAAQLKFRTSKK-NH2 3076 VIVFTFRHNKLIGYERRY 4JBT0050 JBT0050 Ac-SGRGCTKVIVFTFRHNKLIGYERRYNCTS-NH2 3047 JBT0054JBT0050 Biotinyl-Ttds-SGRGCTKVIVFTFRHNKLIGYERRYNCTS-NH2 3002 JBT0058JBT0050 Ac-SGRGCTKVIVFTFRHNKLIGYERRYNCTS-Ttds-Lysin(biotin)-NH2 3003JBT0129 JBT0050 Ac-SGRG[CTKVIVFTFRHNKLIGYERRYNC]TS-NH2 3026 JBT0130JBT0050 Biotinyl-Ttds-SGRG[CTKVIVFTFRHNKLIGYERRYNC]TS-NH2 3001 JBT0205JBT0050 Ac-KKSGRGCTKVIVFTFRHNKLIGYERRYNCTSKK-NH2 3029 JBT0208 JBT0050Ac-KKSGRGCTKVIVFTFRHNKLIGYERRYNKK-NH2 3027 JBT0211 JBT0050Ac-KKGCTKVIVFTFRHNKLIGYERRYNCTSKK-NH2 3032 JBT0212 JBT0050Ac-KKKVIVFTFRHNKLIGYERRYNCTSKK-NH2 3033 JBT0217 JBT0050Ac-KKTKVIVFTFRHNKLIGYERRYKK-NH2 3062 JBT0218 JBT0050Ac-KKKVIVFTFRHNKLIGYERRYNKK-NH2 3063 JBT0219 JBT0050Ac-KKVIVFTFRHNKLIGYERRYNCKK-NH2 3030 GVWQTHPRYFWTMWPDIKGEVIVLFGT 5JBT0101 JBT0101 Ac-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTSKK-NH2 3036 JBT0052JBT0101 Biotinyl-Ttds-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTSKK-NH2 3004 JBT0103JBT0101 Ac-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTS-Ttds-KK- 3005Lysin(biotinyl)-NH2 JBT0178 JBT0101Ac-KKSGVWQTHPRYFWTMWPDIKGEVIVLFGTKK-NH2 3028 JBT0182 JBT0101Ac-KKGVWQTHPRYFWTMWPDIKGEVIVLFGTSKK-NH2 3037 KWFCGMRDMKGTMSCVWVKF 6JBT0120 JBT0120 Ac-SGASRYKWF[CGMRDMKGTMSC]VWVKFRYDTS-NH2 1047 JBT0124Biotinyl-Ttds-SGASRYKWF[CGMRDMKGTMSC]VWVKFRYDTS-NH2 1290 JBT0247 JBT0120Ac-SGASRYKWFCGMRDMKGTMSCVWVKFRYDTS-NH2 1213 JBT0248 JBT0120Ac-KKSGASRYKWF[CGMRDMKGTMSC]VWVKFRYDTSKK-NH2 1001 JBT0251 JBT0120Ac-KKKWFCGMRDMKGTMSCVWVKFKK-NH2 1202 JBT0252 JBT0120Ac-KKCGMRDMKGTMSCVWVKFRYDKK-NH2 1215 ASFPLAVQLHVSKRSKEMA 7 JBT0122JBT0122 Ac-SGYASFPLAVQLHVSKRSKEMALARLYYKTS-NH2 2002 JBT0126 JBT0122Biotinyl-Ttds-SGYASFPLAVQLHVSKRSKEMALARLYYKTS-NH2 2498 JBT0221 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMALARLYYKTSKK-NH2 2003 JBT0224 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMALARLYYKK-NH2 2298 JBT0225 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMALARKK-NH2 2128 JBT0226 JBT0122Ac-KKSGYASFPLAVQLHVSKRSKEMAKK-NH2 2299 JBT0228 JBT0122Ac-KKASFPLAVQLHVSKRSKEMALARLYYKTSKK-NH2 2016 JBT0232 JBT0122Ac-KKGYASFPLAVQLHVSKRSKEMKK-NH2 2303 JBT0233 JBT0122Ac-KKYASFPLAVQLHVSKRSKEMAKK-NH2 2304

This example provides exemplary methods of generating and characterizingTFPI-inhibitory peptides. All peptides in Table 5 were found to bindhuman TFPI-1α. Mutation analysis demonstrated that at least one aminoacid in a TFPI-binding peptide may be substituted while retainingaffinity for TFPI. The peptides of Table 5 tested in ELISA assays boundTFPI-1a with an EC₅₀ of less than 10 μM (1×10⁻⁵ M) and an IC₅₀ of lessthan 50 μM.

Example 2

Selected TFPI-binding peptides were further characterized in terms of“anti-target” binding. This example demonstrates that TFPI-inhibitorypeptides exhibit reduced affinity for non-TFPI-1 proteins.

TFPI-2 was selected as an anti-target because of its similarity toTFPI-1. The binding kinetics of TFPI-binding peptides to human TFPI-1(residues 29-282 fused at the C-terminus to a 10 His-tag; MW 41 kDa (R&DSystems, Minneapolis, Minn.; catalog number 2974-PI)) murine TFPI-1(residues 29-289 fused at the C-terminus to a 10 His-tag; MW 41 kDa (R&DSystems; catalogue number 2975-PI)), and TFPI-2 (R&D Systems,Minneapolis, Minn.) were studied using a BIAcore 3000™ surface plasmonresonance assay (GE Healthcare, Chalfont St. Giles, UK). TFPI proteinswere immobilized on a C1 chip (GE Healthcare, Order Code: BR-1005-40) byamine coupling chemistry aiming for 500 RU. Several TFPI-bindingpeptides were employed as analytes for interacting with the immobilizedTFPI proteins. A flow rate of 30 μl/min was utilized. After 180 seconds,180 μl of peptide solution was injected at six different concentrationsranging from 3.84 nM to 656.25 nM, followed by a dissociation time of480 seconds. The chip was regenerated with 45 μl 10 mM NaOH. Eachbinding experiment was preceded and followed by four measurements withHBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20) plus 1% DMSOand 0.8% P80. BTAevaluation® Version 4.1 software (GE Healthcare) wasemployed to analyze the data. Sensorgrams were fitted to a 1:1 Langmuirbinding curve to determine k_(on) and k_(off) and calculate K_(D).

Certain tested peptides, e.g., JBT0050, JBT0121, JBT0205 and JBT0211,bound to the blank cell and binding constants from those sensorgramscould not be determined. JBT0133 showed weak binding to TFPI-1.Sensorgrams from other peptides gave reliable binding constants. Resultsfrom BIAcore analysis of several TFPI-inhibitory peptides is provided inTable 6 and FIGS. 19A-19B, and 20-21. Each of the peptides listed inTable 6 presented a K_(D) of less than 10 μM. In addition to thepeptides listed below, JBT0375 and JBT0477, substitution mutants ofJBT0293 at amino acid position 5 (JBT0375) or amino acid positions 5 and10 (JBT0477), also exhibited a K_(D) of less than 10 μM. Sensorgrams oftwo of the peptides are provided as FIGS. 9A and 9B.

TABLE 6 Peptide k_(on) (1/Ms) k_(off) (1/s) K_(D) (M) JBT0047 4.0 × 10⁵1.9 × 10⁻² 4.7 × 10⁻⁸ JBT0120 1.17 × 10⁶  4.78 × 10⁻²  4.08 × 10⁻⁸ JBT0131 1.4 × 10⁵ 6.0 × 10⁻² 4.31 × 10⁻⁷  JBT0132 3.55 × 10⁴  3.26 ×10⁻²  9.17 × 10⁻⁷  JBT0224 6.39 × 10⁴  1.95 × 10⁻²  3.05 × 10⁻⁷  JBT02936.0 × 10⁵ 5.6 × 10⁻² 9.5 × 10⁻⁸ JBT0297 5.0 × 10⁵ 1.4 × 10⁻² 2.9 × 10⁻⁸JBT0303 8.13 × 10⁵  2.75 × 10⁻²  3.4 × 10⁻⁸ JBT0305 7.5 × 10⁵ 3.1 × 10⁻²6.1 × 10⁻⁸

Interaction with the TFPI-2 anti-target also was examined. The maximumsignal generated from candidate peptide interaction with human TFPI-2was much lower than the signals obtained with TFPI-1 as an interactionpartner. Kinetic analysis of the low TFPI-2 binding signals was prone toerror; therefore, visual comparison of sensorgrams was used to estimatebinding affinity. A sensorgram illustrating JBT0120 binding to TFPI-1and TFPI-2 is provided as FIGS. 10A and 10B. JBT0120 binds TFPI-2 with10-fold lower affinity compared to its binding affinity for TFPI-1.JBT0132 also was found to exhibit at least 10-fold greater affinity forTFPI-1 than TFPI-2.

The data provided by this example confirm that TFPI-inhibitory peptidesspecifically bind TFPI-1.

Example 3

The following example describes the characterization of TFPI-inhibitoryactivity of select peptides identified in Example 1 using FXa inhibitionand extrinsic tenase inhibition assays. Both assays are predictive ofactivity in plasmatic systems. The extrinsic tenase assay gives insightinto the influence of the peptides on (a) the interaction of FXa andTFPI and (b) the interaction of the FXa-TFPI complex with the TF-FVIIacomplex. The FXa inhibition assay measures a peptide's influence on theinteraction of FXa and TFPI only.

The extrinsic tenase complex is responsible for FX and FIX activationupon initiation of the coagulation process. The extrinsic complex iscomposed of FVIIa, Tissue Factor (TF), and FX substrate. To determinethe influence of peptides on the TFPI-mediated inhibition of theextrinsic tenase complex, a coupled enzyme assay was established.Peptides were diluted 1/6.25 from 10 mM stocks (in DMSO) and furtherdiluted by serial 1/4 dilutions in buffer or DMSO to prevent unwantedprecipitation. TFPI was diluted in HNaCa-HSA or BSA (25 mM HEPES; 175 mMNaCl; 5 mM CaCl₂; 0.1% HSA or BSA; pH 7.35). FVIIa, lipidated TF,phospholipid vesicles (DOPC/POPS 80/20), and chromogenic substratespecific for FXa (S-2222 (available from DiaPharma, West Chester,Ohio)), all diluted in HNaCa-HSA, were added to 96-well plates. After anincubation period, TFPI and peptide dilutions were added, resulting in afinal concentration of 2.5% DMSO (if present in the peptide stock). FXactivation was initiated by adding FX to the wells. FXa-mediatedchromogenic substrate conversion was determined by observing an increasein absorbance using a micro-plate reader. The amount of FXa generated atcertain time points was calculated from the OD readings. FXa generatedat 20 minutes after start of the reaction was considered for calculationof EC₅₀ from plots of peptide concentration versus the inhibition ofTFPI (%).

The functional inhibition of TFPI also was examined using a FXainhibition assay. A FXa-specific chromogenic substrate (S-2222) andTFPI, both diluted in HNaCa-HSA, were added to 96 well plates. Peptideswere diluted 1/6.25 from 10 mM stocks (in DMSO or Aqua-Dest) and furtherdiluted by serial 1/4 dilutions in buffer or DMSO to prevent unwantedprecipitation. The peptide dilutions (2.5 μl) were added to the 96 wellplates, resulting in a final concentration of 2.5% DMSO (if present inthe peptide stock). The conversion of chromogenic substrate wastriggered by the addition of FXa, and the kinetics of the conversionwere measured in a micro-plate reader. Because TFPI inhibits FXa slowly,OD readings after 115 minutes were considered for calculation of theEC₅₀ from plots of peptide concentration versus the inhibition of TFPI(%).

Results from the extrinsic tenase assay and FXa inhibition assay areprovided in Table 7 and FIGS. 22A-22M, 23A-23L, 24A-24D, 25A-25L,26A-26F and 27A-27C.

TABLE 7 FXa Inhibition Assay Extrinsic Tenase Assay % inhibition %inhibition EC₅₀ [μM] @ 2.5 μM EC₅₀ [μM] @ 2.5 μM JBT0120 0.9 45 0.9 45JBT0132 1.2 36 0.1 10 JBT0224 n.a. 26 3.5 18 JBT0303 1.2 61 n.a. 8

Referring to Table 7, JBT0120, JBT0132, and JBT0224 restored extrinsiccomplex-mediated FX activation in the presence of TFPI-1 with an EC₅₀ of<2 μM, resulting in between about 20% to about 60% inhibition of TFPIactivity. JBT0047 (EC₅₀=1.4 μM), JBT0131 (EC₅₀=2.2 μM), and JBT0293(EC₅₀=2.9 μM) also restored extrinsic complex activity in the presenceof TFPI-1. In addition, JBT0120, JBT0132, JBT0224, and JBT0303 restoredFXa activity in the presence of TFPI-1 with an EC₅₀ of <5 μM, resultingin between about 5% to about 50% inhibition of TFPI activity, in the FXainhibition assay. JBT0047 (EC₅₀=0.7 μM), JBT0131 (EC₅₀=8.2 μM), JBT0293(EC₅₀=1.3 μM), JBT0297 (EC₅₀=0.6 μM), and JBT0305 (EC₅₀=2.3 μM) alsorestored activity of FXa in the presence of TFPI-1 in the FXa inhibitionassay. This example confirms that peptides of the invention are TFPIantagonists.

Example 4

In this example, the TFPI inhibitory activity of peptides is establishedusing a plasma-based assay.

The influence of peptides on thrombin generation was measured induplicate via calibrated automated thrombography in a Fluoroskan Ascent®reader (Thermo Labsystems, Helsinki, Finland; filters 390 nm excitationand 460 nm emission) following the slow cleavage of thethrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC (Hemker,Pathophysiol. Haemost. Thromb., 33, 4-15 (2003)). Plasma from patientswith FVIII or FIX deficiency (George King Bio-Medical Inc., OverlandPark, KN) was obtained for testing. The residual coagulation factoractivity for each of the plasmas was lower than 1%. As a model forantibody-mediated FVIII deficiency, frozen pooled normal plasma (GeorgeKing Bio-Medical Inc., Overland Park, KN) was incubated with high titer,heat inactivated, anti-human FVIII plasma raised in goat (4490 BU/ml;Baxter BioScience, Vienna, Austria) giving rise to 50 BU/mL. The plasmaswere mixed with corn trypsin inhibitor (CTI) (Hematologic Technologies,Inc., Essex Junction, Vt.) to inhibit Factor XIIa contamination,resulting in a final concentration of 40 μg/mL.

Pre-warmed (37° C.) plasma (80 μL) was added to each well of a 96 wellmicro-plate (Immulon 2HB, clear U-bottom; Thermo Electron, Waltham,Mass.). To trigger thrombin generation by Tissue Factor, 10 μL of PPPlow reagent containing low amounts (12 pM) of recombinant human TissueFactor and phospholipid vesicles composed of phosphatidylserine,phosphatidylcholine and phosphatidylethanolamine (48 μM) (ThrombinoscopeBV, Maastricht, The Netherlands) were added. Peptides were diluted 1/7.5from 10 mM stocks with DMSO, and further diluted 1/8.33 with Aqua-Destresulting in a DMSO concentration of 12%, providing a 0.5% DMSOconcentration in the final assay mix. Just prior putting the plate intothe pre-warmed (37° C.) reader, 5 μL of HEPES buffered saline with 5mg/mL human serum albumin (Sigma-Aldrich Corporation, St. Louis, Mo.,USA) or 12% DMSO in Aqua-Dest was added, followed by addition of thepeptide dilutions or reference proteins (FVIII Immunate referencestandard (Baxter BioScience, Vienna, Austria); Factor VIII InhibitorBy-Passing Activity (FEIBA) reference standard (Baxter BioScience,Vienna, Austria); NovoSeven (Novo Nordisk, Denmark); and purified humanplasma FIX (Enzyme Research Laboratories, South Bend, Ill.)). Thrombingeneration was initiated by dispensing into each well 20 μL of FluCareagent (Thrombinoscope BV, Maastricht, The Netherlands) containing afluorogenic substrate and HEPES-buffered CaCl₂ (100 mM). Fluorescenceintensity was recorded at 37° C.

The parameters of the resulting thrombin generation curves werecalculated using Thrombinoscope™ software (Thrombinoscope BV,Maastricht, The Netherlands) and thrombin calibrator to correct forinner filter and substrate consumption effects (Hemker, Pathophysiol.Haemost. Thromb., 33, 4-15 (2003)). For calculating the thrombingenerating activity of certain peptide concentrations equivalent to thereference proteins (e.g., FVIII Immunate® reference standard, FEIBAreference standard), the thrombin amounts at the peak of each thrombingeneration curve (peak thrombin, nM) were plotted against the standardconcentrations, and fitted by a non-linear algorithm. Based on thiscalibration, FVIII Immunate, FIX, FEIBA or NovoSeven equivalentactivities were calculated. Results for various peptides are provided inFIGS. 12A-12P, 13A-13D, 14A-14E, 15A-15D, 16A-16F, 17A-17B, 18A-18E,28A-28N, 29A-29F and 30A-30D. Representative results are provided inTable 8. (* denotes that FVIII deficient plasma was obtained from adifferent donor.)

TABLE 8 % FVIII-equivalent activity FEIBA-equivalent activity in inFVIII deficient plasma @ FVIII inhibited plasma @ 10 μM peptide 10 μMpeptide [mU/ml] JBT0120 37.4* 298 JBT0132 5.3 41 JBT0224 16.2 191JBT0303 20.8 253

With reference to Table 8, JBT0120, JBT0132, JBT0224, and JBT0303improved TFPI-dependent thrombin generation in FVIII-depleted plasma tolevels exceeding 1% of the level of thrombin generation in plasmacontaining FVIII (% FVIII-equivalent activity). The tested peptidesexhibited approximately 5%-40% FVIII-equivalent activity inFVIII-deficient plasma. JBT0120 and JBT0132 improved peak thrombin andpeak time, dose dependently, as illustrated in FIGS. 11A and 11B.

Substitution mutants based on the amino acid sequence of JBT0293 alsowere tested in a plasma-based assay, as well as the FXa inhibition andextrinsic tenase inhibition assay described in Example 3. Representativeresults are provided in Table 9.

TABLE 9 FVIII- equivalent Extrinsic activity FXa Tenase (mU/ml) inBiacore Inhibition Inhibition Hem A K_(D) EC₅₀ EC₅₀ plasma @ 1 μM (nM)(μM) (μM) peptide JBT0047 47 0.7 1.4 45 JBT0293 97 1.3 2.9 48 JBT0303 341.2 NA 125 JBT0500 8.2 0.12 — 372 JBT0740 2.4 0.07 — 333 JBT1584 0.30.01 — 489

Additionally, JBT0477, which comprises the amino acid sequence ofJBT0293 but for substitutions at amino acid positions 5 and 10 of theJBT0293 sequence, improves thrombin generation equivalent to 413 mU/mlof FVIII (at 1 μM of peptide) in FVIII-deficient plasma. Substitutionmutation of JBT0293 resulted in highly optimized peptides with respectto affinity for TFPI and improved activity in FXa inhibition, extrinsictenase inhibition, and plasma-based assays.

Example 5

The following example demonstrates that the peptides of the inventioncan be modified by the addition of moieties that enhance physicochemicalor pharmacokinetic properties of the peptides. As illustrated below, theaddition of 40 kDa PEG to peptides described herein dramaticallyimproved the pharmacokinetic behavior of the peptides. The example alsodescribes optimization of a TFPI-binding peptide, JBT1857, to reducesusceptibility to proteolysis.

Methods of conjugating chemical or biological moieties to peptides areknown in the art. To add PEG (polyethylene glycol) to the peptidesdescribe herein, a functional group (AOA=aminooxy acetate) was added tothe N-terminus of the peptides for coupling to aldehydes and ketones.Alternatively, a cysteine was added to the C-terminal part of thepeptide for coupling with maleimid (Hermanson, Bioconjugate Techniques,Academic Press (1996)). The peptides (JBT1586)AOA-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 166) and (JBT1587)Ac-FQSKGNVFVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 167) were used forN-terminal and C-terminal modification with PEG, respectively.AOA-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 166) andAc-FQSKGNVFVDGYFERL-Aib-AKLC-NH2 (SEQ ID NO: 167) were incubated withexcess 40 kDa mPEG-Propionaldehyde (SUNBRIGHT ME-400AL2, NOF, Japan) and40 kDa mPEG-maleimide (SUNBRIGHT ME-400MA, NOF, Japan), respectively.The resulting PEGylated peptides, JBT1852 and JBT1855, show similaraffinities compared to the starting structureAc-FQSKGNVFVDGYFERL-Aib-AKL-NH2 (JBT0740) (SEQ ID NO: 66).

The resulting PEGylated peptides demonstrated significantly increasedplasma stability and prolonged plasma half-life in mice. FIG. 31illustrates the results from a pharmacokinetic analysis of the freepeptide JBT0740 (Ac-FQSKGNVFVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 66)compared to the C-terminally PEGylated peptide JBT1855(Ac-FQSKGNVFVDGYFERL-Aib-AKLC(PEG(40 kD))-NH2) (SEQ ID NO: 252)following intravenous administration to mice. In contrast to theunPEGylated peptide, the PEGylated peptide is present at highconcentrations in mouse plasma at 100 minutes post-administration. TheunPEGylated peptide is rapidly cleared from the plasma. FIG. 40illustrates the results from a pharmacokinetic analysis of JBT1855following subcutaneous injection. JBT1855 also strongly improvedthrombin generation in the assay described in Example 4 (FIG. 41).

The JBT1852 and JBT1855 peptides also were characterized in the assaysdescribed in Examples 1-4 and compared to JBT0740 and other peptides inthe JBT0047 family. Representative results are provided in Table 10 setforth below.

TABLE 10 FVIII-equivalent activity TFPI-1α FXa (mU/ml) in SolubilityAffinity (nM) Inhibition FVIII deficient (mg/ml; PBS Plasma StabilityBiacore IC₅₀ plasma @ 1 without (half life in minutes) K_(D) (μM) μMpeptide Ca²⁺ and Mg²⁺) mouse human JBT0717 1.1 0.05 421 0.97 24 >120JBT0740 2.4 0.06 333 0.92 50 >120 JBT1584 0.3 0.02 486 2.66 40 >120JBT1852 11.1 0.17 >1000 >1.00* >120 >120 JBT1855 10.50.07 >1000 >1.00* >120 >120 *formulated in 25 mm HEPES, pH 7.35, 175 mMNaCl

The peptides listed in Table 10 also were assayed for interaction withthe TFPI-2 anti-target, and generated signals too low for reliableaffinity measurement. The data suggest that PEGylation does not ablatethe inhibitory activity of the inventive peptides or negatively affectselectivity for TFPI-1.

Cell-Based Extrinsic Tenase Assay

The ability of the TFPI-binding peptides described above to restoreextrinsic tenase complex-mediated conversion of FX to FXa also wasdetermined using a cell-based extrinsic tenase assay. The cell-basedextrinsic tenase assay also was employed to explore the influence ofPEGylation on an exemplary TFPI-binding peptide of the invention,JBT0740. Human umbilical vein endothelial cells (HUVEC) were counted andseeded in complete growth medium in a 96-well plate (black flat withclear bottom) at a density of 1.5×10⁴ cells per well. Cells were grownovernight (for approximately 16 to 18 hours), washed twice withpre-warmed basal medium, stimulated with 1 ng/ml recombinant TNFα (SigmaAldrich (Cat. No. T6674)) in 200 μl of basal medium for four hours at37° C., and washed twice with 200 μl of pre warmed cell culture buffer.Buffer (50 μl) containing FVIIa (Enzyme Research Laboratories),TFPI-binding peptides (dissolved in either DMSO or Hepes buffered salinewith or without 0.1% Tween-80), or αTFPI antibodies were applied to thecells and incubated for 20 minutes at 37° C., allowing FVIIa/TF complexformation and binding of TFPI antagonists to TFPI. After the incubationperiod, 50 μl of cell culture buffer containing FX and a FXa-specificsubstrate (Fluophen FXa (HYPHEN BioMed)) was applied, resulting in afinal volume of 100 μl cell culture buffer mix on the cells. The finalconcentrations were: 39 μM FVIIa; 170 nM FX; 250 μM Fluophen FXa, and2.5% DMSO (when peptides were dissolved in DMSO).

The 96 well plate was transferred to a pre-warmed (37° C.) fluorescencereader for detecting FXa-specific fluorogenic substrate conversion byFXa, which is generated by the TF/FVIIa complex on the surface ofstimulated HUVECs. Readings taken after nine minutes of incubation wereused for calculation of the TFPI inhibitory effect of the TFPI-bindingpeptides or antibodies. The approximate percent inhibition of TFPIobserved at various concentrations of the following peptides (belongingto the JBT0047 family) is set forth in Table 11: JBT0717(Ac-FQSK-Nmg-NVFVDGYFERLRAKL-NH2) (SEQ ID NO: 61), JBT0740(Ac-FQSKGNVFVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 66), JBT1584(Ac-FQSK-Nmg-NVFVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 164), and JBT1857(Ac-FQSKpNVHVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 178).

TABLE 11 % TFPI inhibition 40 μM 8 μM 1.6 μM 0.32 μM 64 nM JBT0717 60%50% 32% 28% 20% JBT0740 70% 39% 28% 14%  3% JBT1584 73% 62% 51% 40% 29%JBT1857 80% 57% 41% 35% 15%

PEGylated peptides also were tested using the cell-based extrinsictenase assay. JBT0740 (SEQ ID NO: 66) was conjugated to a 1 kD PEGmoiety at the N-terminus to produce JBT1853 or at the C-terminus toproduce JBT1854. JBT1853 and JBT1854 inhibited TFPI by 20% or lessdepending on the amount of peptide used in the assay. JBT1855, whichcomprises a 40 kD PEG moiety at the C-terminus (parent peptide, JBT0740)performed better in the cell-based assay than JBT1852, which comprises a40 kD PEG moiety at the N-terminus. JBT1855 mediated 20-30% TFPIinhibition, while JBT1852 inhibited TFPI activity by 10% or less.

Peptides of the JBT0120 family, JBT0120, JBT0415, JBT0444, JBT1426, andJBT1837, also were tested in the cell-based extrinsic tenase assay andfound to inhibit TFPI to a lesser degree compared to peptides of theJBT0047 family. The reduced or partial inhibitory activity may bedesired in some embodiments of the invention. Similar to the peptides ofthe JBT0047 family, peptide optimization increased TFPI inhibitoractivity of JBT0120 family peptides.

In the course of examining the stability and inhibitory activity ofJBT1857, it was determined that the amino acid sequence of the peptidecontained a protease cleavage site between Val9 and Asp10. Substitutionof Tle at position 9 (generating JBT2431) and substitution of Pro atposition 10 (generating JBT2432) blocked cleavage of the peptide andenhanced the plasma stability of the peptide by about three-fold from27% (JBT1857) to 82% (JBT2431) and 76% (JBT2432). An additional putativecleavage site was identified between Gly11 and Tyr12. A G11asubstitution (generating JBT2414) further improved the stability of thepeptide to 100%. All stabilities were determined by quantitative ELISAafter 24 hour incubation in human plasma.

The results described above demonstrate that optimization of theTFPI-binding peptides described herein utilizing non-conventional aminoacids improved TFPI inhibition and plasma stability. Additionally,PEGylated peptides of the invention inhibit TFPI activity in acell-based extrinsic tenase assay, with C-terminal PEGylated peptidesperforming better than N-terminal PEGylated peptides. The TFPI-bindingpeptides of the invention inhibit the activity of both free TFPI andcell-bound TFPI.

Example 6

The following example illustrates the ability of peptides describedherein to reduce bleeding in an animal model.

Ten week old C57B1/6NCrl mice were housed for two weeks prior to thestudy. Thirty minutes before the nail clip, the animals wereadministered (a) JBT1855 (10 mg/kg) intravenously (i.v.) via the tailvein or subcutaneously (s.c.) in the neck region, (b) anti-TFPI antibody(18 mg/kg; i.v.), or (c) vehicle (175 mM NaCl, 25 mM HEPES, pH 7.35; 10ml/kg; i.v.). The animals were anaesthetized with 80 mg/kg pentobarbitalten minutes prior to the nail clip. To achieve bleeding, the nail of thesmall toe of the right hind paw was removed. The paw was submerged in a0.9% NaCl solution for blood collection for a period of 60 minutes.Blood loss was quantified after lysis by spectrophotometry. Thetemperature was kept constant at 37° C. over the course of theexperiment. The results of the study are illustrated in FIG. 42 andsummarized in Table 11.

TABLE 11 JBT1855 JBT1855 α-TFPI Vehicle i.v. s.c. i.v. i.v. Mean (in μl)29.9 31.7 1.9 74.9 (SD) (71.4) (31.5) (1.4) (74.6) # of mice 12 12 12 12p-value 0.07 0.04 0.001

Intravenous or subcutaneous administration of JBT1855, a PEGylatedpeptide of the invention, reduced blood loss in mice compared totreatment with vehicle alone.

Example 7

The following example describes characterization of TFPI-peptideinteractions via nuclear magnetic resonance and x-ray crystallography.In particular, the TFPI binding site of the antagonistic peptidesJBT0303, JBT0122 and JBT0415; the residues of JBT0303, JBT0122 andJBT0415 interacting with TFPI160; and the secondary structure ofcomplexed and free JBT0303, JBT0122 and JBT0415 were investigated at amolecular level using 2D ¹⁵N-heteronuclear single quantum coherence(HSQC) spectra. The interaction of JBT1857 and KD 1 of TFPI was examinedusing x-ray crystallography, and the residues of TFPI KD 1 that mediateJBT1857 binding were mapped.

Identification of the Binding Site of JBT0303 on TFPI160

A ¹⁵N-labelled preparation of TFPI160 was used for titration experimentsof TFPI160 with JBT0303. HSQC spectra of a ˜500 μM ¹⁵N-TFPI160 samplewithout and with increasing amounts of peptide were recorded at 30° C.on a Varian 600 MHz spectrometer. The peptide-protein interaction showedslow exchange behavior (k_(ex)<<Δω), meaning that each TFPI residueresults in a defined signal for the free protein and the protein-peptidecomplex. Unlike fast exchange behavior (k_(ex)>>Δω), where a mixtureresults in only one peak with averaged position according to thepopulation of the species, slow exchange behavior does not allowtracking of the signals upon peptide binding. Thus, in order to locatethe binding site, the shifted peaks of the TFPI160-JBT0303 complexneeded to be assigned. This required the preparation of a sample of¹³C/¹⁵N-TFPI160 and JBT0303.

Initially, a sample was prepared with 992 μM ¹³C/¹⁵N-TFPI160 and 1190 μMJBT0303. However, the NMR sample resulted in poor quality spectra whichdid not allow assignment of the complex. The sample gelled, likely dueto the formation of high molecular weight aggregates. Thus, the acquiredNMR data predominantly showed signals arising from the most flexibleparts of the isotope-labeled TFPI160. Therefore, sample conditions werereinvestigated for further experiments. From a series of ¹⁵N-HSQCexperiments conducted on the TFPI160-JBT0303 complex, it was concludedthat gel formation could be avoided by sample dilution and dataacquisition at elevated temperature. The final concentration of¹³C/¹⁵N-TFPI160 was 331 μM and that of JBT0303 was 397 μM. Spectraquality was improved. Due to the lower concentration and reducedsignal-to-noise ratio, assignment had to be performed based on HNCA,HNCO and HNCOCA experiments.

Except for four previously assigned residues, all residues that could beassigned in the apo-TFPI160 could be assigned in the TFPI160-JBT0303complex. Assignment of some residues was ambiguous due to the lack ofpeaks in the 3D spectra. Furthermore, the peaks of three residues wereonly visible in the HSQC spectrum from the original titrationexperiment. However, all peaks in the vicinity where unambiguouslyassigned and, therefore, the assignment of these residues is likely tobe correct.

Chemical shift changes of the HSQC signals of ¹⁵N-TFPI160 bound toJBT0303 compared to free TFPI160 is illustrated in FIG. 43. Residuesundergoing the strongest chemical shift were exclusively on Kunitzdomain 1. Chemical shifts of residues F25, F28, D32, A37, T48 and Y56shifted the most (>2 ppm). Residues I38, I46, F47 and F54 also shiftedmore than 1.5 ppm. It is unclear whether residues N-terminal of F25 areinvolved the interaction with JBT0303 because residues 20-24 are notassigned. L19 shows a change of chemical shift amounting to ˜0.6 ppm.Thus, in contrast to previous beliefs that amino acids within residues1-18 of TFPI are involved in peptide binding, the present data suggestthat there is little, if any, peptide binding to the N-terminal tail ofTFPI. A ribbon model of the secondary structure of TFPI proteinillustrating regions of chemical shift changes of HSQC signals ofTFPI160 bound to JBT0303 compared to free TFPI160 is set forth in FIG.44.

To more particularly identify the binding site of JBT0303 on TFPI160,the amide exchange rates of ¹⁵N-TFPI160 and ¹⁵N-TFPI160+JBT0303 weredetermined. The amide exchange experiment mainly detects changes in theenvironment of the peptide backbone by measuring H exchange of amidegroups. The H₂O frequency is irradiated with a power high enough that itis not dissipated by relaxation, resulting in a complete saturation andsuppression of the H₂O signal. A side effect of this method of H₂Osignal suppression is that the suppression is transferred toexchangeable amide NHs which exchange with solvent (H/H exchange). Thesaturation transfer is dependent on the H/H exchange rate which issemi-quantitative. The effect is reduced for more protected NH groups(i.e., unprotected NHs are attenuated more than protected NHs). If aprotected NH lies in proximity to H-alphas of a ligand, a higherexchange rate is observed compared to the apo form. Similarly, Hexchanges can be mediated by the OH groups of Ser, Thr or Tyr.

HSQC spectra without and with water suppression of apo ¹⁵N-TFPI160 andthe ¹⁵N-TFPI160-JBT0303 complex were recorded. The relative exchangerate of each residue of TFPI160 was determined by calculating the ratioof the peak intensities in the HSQC spectra with and without watersuppression. A comparison of the data sets of ¹⁵N-TFPI160 and¹⁵N-TFPI160+JBT0303 revealed that TFPI residues 25, 26, 36, 62, 63, 127,132 and 152 exhibited greater than 10% decreased amide exchange rate inthe complex, whereas residues 29, 30, 42, 45, 49, 50, 56, 66 and 98exhibited more than 10% increased exchange rate.

Constraints derived from the amide exchange experiment were included forthe calculation of refined HADDOCK models: (a) torsion angles are takenfrom the calculations of TALOS for K4, K5, V7, F8, Y12-A18 of JBT0303(chemical shift experiments); (b) residues of KD 1 with chemical shiftchanges of more than 1.5 ppm are involved in binding JBT0303: F25, F28,D32, A37, I38, I46, F47, T48, F54 and Y56 (chemical shift experiments);(c) the hydrophobic side of the amphipathic helix of JBT0303 is bound toKD1: Y12 or L16 or L20 of JBT0303 bind to D32 or A37 or 138 or F54 orY56 of KD1 (chemical shift experiments); (d) R15 or K19 of JBT0303 bindto D31 or D32 or E60 of KD1 (chemical shift experiments); (e) F8 or V9of JBT0303 bind to F25 or F28 of KD1 (chemical shift experiments); (f)Y12 or F13 of JBT0303 bind to 146 or F47 or T48 of KD1 (chemical shiftexperiments); (g) Q2 of JBT0303 binds to Y56 of KD1 (chemical shiftexperiments); (h) F1 of JBT0303 binds to M39 or F66 of KD1 (amideexchange experiments); (i) S3 or K4 or K5 of JBT0303 bind to F66 (amideexchange experiments); (j) V7 or F8 or V9 of JBT0303 bind to F25 or C26or N62 or Q63 of KD1 (amide exchange experiments); (k) V9 or D10 or G11or R15 of JBT0303 bind to F28 or K29 or A30 of KD1 (amide exchangeexperiments); (l) Y12 or F13 of JBT0303 bind to N45 of KD1 (amideexchange experiments); (m) Y12 or F13 or E14 or R15 bind to R49 or Q50of KD1 (amide exchange experiments); and (n) L20 of JBT0303 binds to K36of KD1 (amide exchange experiments). The data converged to essentiallyone model of the KD1+JBT0303 complex.

Identification of the Binding Site of JBT0122 on TFPI160

As with the ¹³C/¹⁵N-TFPI160+JBT0303 complex, the ¹³C/¹⁵N-TFPI160+JBT0122NMR sample resulted in spectra of poor quality due to the formation of agel. The concentration of 723 μM ¹³C/¹⁵N-TFPI160+JBT0122 lead toformation of higher order aggregates. The sample was diluted to 361.5 μMand spectra recorded at 37° C., resulting in improved spectra quality.HNCO, HNCA and HNCOCA spectra were acquired. Except for five residues,all of the previously assigned peaks of apo-TFPI160 could be assigned inthe TFPI160-JBT0122 complex. Residues undergoing the strongest chemicalshift changes and likely to interact with the peptide often did notresult in peaks in the 3D spectra. Peaks in the linker region betweenKunitz domain 1 (KD1) and Kunitz domain 2 (KD2), however, also exhibitedlow intensities. Hence, the assignment of these peaks is ambiguous. Somepeaks were only visible in the HSQC of the original titrationexperiment. Their assignment was in most cases certain, as the peaksoverlapped in the TFPI160 and the TFPI160+JBT0122 HSCQ spectra.

Chemical shift changes of the HSQC signals of ¹⁵N-TFPI160 bound toJBT0122 compared to free TFPI160 is illustrated in FIG. 45. Significantchemical shift changes were exclusively found for residues of KD2. Ingeneral, the extent of the chemical shift changes caused by binding ofJBT0122 to TFPI160 was less pronounced than that of JBT0303. Residueswith the strongest perturbation of chemical shift were F96, G128, G129,G132, N133 and N136. C97, E101, T111, F114, N135 and F137 wereperturbed, exhibiting chemical shift changes of more than 0.5 ppm. Aribbon model of the secondary structure of TFPI protein illustratingregions of chemical shift changes of HSQC signals of TFPI160 bound toJBT0122 compared to free TFPI160 is set forth in FIG. 46.

Identification Residues of JBT0122 that Interact with TFPI160

For the sequential backbone signal assignment of JBT0122,¹³C/¹⁵N-labelled peptide was produced recombinantly. Briefly, thepeptide was expressed as a fusion protein with thioredoxin in E. coli.¹³C/¹⁵N-labelled peptide was prepared using M9 medium containing 3.0 g/l¹³C-glucose and 1.0 g/l ¹⁵NH₄Cl. The fusion protein was affinitypurified using a Ni-chelating column and a poly-histidine tag. Thepeptide was cleaved by thrombin. The thioredoxin/his-tag and thrombinwas removed using a Ni-chelating column and a benzamidine column,respectively. The peptide was then purified by reverse phasechromatography. Purity, integrity, and identity were verified bySDS-PAGE, RP-HPLC and mass spectrometry. Recombinant JBT0122 was namedJBT0788 and had two additional residues at its N-terminus, glycine andserine, which represent the remains of the thrombin cleavage site.

The assignment of JBT0788 was done on the basis of HSQC, HNCACB, HNCA,HNCO and HNN spectra recorded at 10° C. on a Varian 600 MHz spectrometerand assigned using the SPARKY software. The temperature was reducedcompared to NMR experiments with TFPI160 to improve spectra quality.From the recorded spectra, the carbonyl carbon (C), the alpha carbon(CA), the beta carbon (CB), the amide proton (H), and the amide nitrogen(N) of most residues were assigned. The assignment for residues H13 andR17 was ambiguous. An HNCOCA led to an unambiguous assignment for theseresidues.

An assignment table for JBT0788 is provided in FIG. 47. Two sets ofsignals for residues 4-12 were observed in the spectra of JBT0788.Considering that the primary structure of JBT0788 is not compromised,the two sets of signals likely result from a cis/trans isomerization ofthe peptide bond between F6 and P7. A ratio of 76:24 was determined formajor:minor conformation based on the intensities of the correspondingsignals in the HSQC spectrum. As judged from the Cα shift of theproline, the major conformation is likely trans, as its Cα value of63.16 ppm is higher than of the minor conformation (62.49 ppm).

One purpose of the assignment was to extract the secondary structure ofthe peptide from Cα chemical shifts. Cα chemical shifts are influencedby the angles φ and ψ and, thus, by the secondary structure of thepeptide. In β-strands, Cα are generally shifted to lower ppm; inα-helices, Cα are generally shifted to higher ppm. By subtracting themeasured Cα value from a tabulated random coil value, negative valuesare calculated for residues in β-strands and positive values forresidues in α-helices. Thus, a batch of consecutive negative valuesindicates a β-strand while a batch of consecutive positive valuesindicates an α-helix.

JBT0788 exhibited a broad patch of increased Cα values(Δδ(Cα)=Cα_(measured)−Cα_(random coil)) indicating an α-helix comprisingresidues 8 to 26. Δδ(Cα) values for stable a-helices within tertiarystructures of native proteins are typically between 3-4 ppm. Δδ(Cα)values of the α-helix of JBT0788 rise up to about 1.7 ppm, indicatingmore flexibility than an average helix within a protein. Another featureof JBT0788 is the proline at position 7, directly N-terminal to theα-helix, which fits well as α-helices in proteins are frequentlyterminated by a proline at the N-terminus. Residue 6 has a strongnegative value, which is caused by the neighboring proline known toforce its N-terminal neighbor into a β-strand-like conformation. Thestrong positive value of C-terminal residue 31 is also typical forresidues without a C-terminal neighbor. The peptide bond between F6 andP7 in JBT0788 adopts two conformations, a trans (76%) and a cisconformation (24%). The conformation at this position impacts theconformation of the consecutive residues. In the trans isoform, thea-helix starts immediately after P7; the α-helix of the cis isoform doesnot start until residue L12. A ribbon model illustrating the secondarystructure of free JBT0788 is set forth in FIG. 48.

The chemical shifts within JBT0788 can also be employed to calculate thetorsion angles using TALOS software. TALOS is a database system forempirical prediction of φ and ψ backbone torsion angles using acombination of five kinds (HA, CA, CB, CO, N) of chemical shiftassignments for a given protein or peptide sequence. The TALOS approachis an extension of the observation that many kinds of secondary chemicalshifts (i.e., differences between chemical shifts and theircorresponding random coil values) are correlated with aspects of proteinsecondary structure. The goal of TALOS is to use secondary shift andsequence information in order to make quantitative predictions for theprotein backbone angles φ and ψ, and to provide a measure of theuncertainties in these predictions. TALOS uses the secondary shifts of agiven residue to predict φ and ψ angles for that residue. TALOS alsoincludes the information from the next and previous residues when makingpredictions for a given residue. The idea behind TALOS is that if onecan find a triplet of residues in a protein of known structure withsimilar secondary shifts and sequence to a triplet in a target protein,then the φ and ψ angles in the known structure will be useful predictorsfor the angles in the target. In practice, TALOS searches a database forthe 10 best matches to a given triplet in the target protein.

In order to assign the HSQC spectrum of JBT0788 complexed with TFPI160,a sample consisting of 400 μM ¹³C/¹⁵N-JBT0788 and 400 μM TFPI160 wasprepared. As with previous NMR samples of peptide and TFPI160, thesample gelled. The sample was diluted and the pellet dissolved indeuterated DMSO, resulting in a final concentration of ˜300 μM¹³C/¹⁵N-JBT0788+TFPI160 and 5% DMSO. Measurements were performed at 40°C. This improved the quality of the acquired spectra. Experiments wereacquired in the TROSY mode to account for the relaxation properties of apartially aggregated sample. Cryo-probe technology on the Varian 600 MHzspectrometer was employed due to the low concentration of theprotein-peptide complex in the sample. The resulting data quality wassufficient to obtain the backbone shifts of JBT0788 when utilizing thecryo-probe technology and acquiring the triple-resonance experiments induplicate. The assignment of JBT0788 in complex with TFPI160 wasperformed on the basis of HNCA, HNCOCA and HNCO spectra. From therecorded spectra, the carbonyl carbon (CO), the alpha carbon (CA), theamide proton (H), and the amide nitrogen (N) of most residues wereassigned. An assignment table for JBT0788 complexed to TFPI160 isprovided in FIG. 49.

A feature of apo-JBT0788 was the presence of two sets of signals foramino acid residues 4-12, likely resulting from a cis/transisomerization of the peptide bond between F6 and P7. In theJBT0788-TFPI160 complex, only one set of peaks is observed, implyingthat only one of the conformations binds to TFPI160. Apo-JBT0788 alsoexhibited a broad patch of increased Cα values(Δδ(Cα)=Cα_(measured)−Cα_(random coil)=positive) indicating an α-helixreaching from residue 8 to residue 26. As mentioned above, Δδ(Cα) valuesfor stable a-helices within tertiary structures of native proteins aretypically between 3-4 ppm. Δδ(Cα) values of the α-helix of apo-JBT0788increase to about 1.7 ppm, indicating more flexibility than an averagehelix within a protein. When complexed with TFPI, residues 8 to 26exhibited values of between 3-5 ppm, indicating the formation of astable α-helix or helices. A ribbon model illustrating the secondarystructure of JBT0788 when complexed with TFPI160 is set forth in FIG.50. Large chemical shift changes within JBT0788 caused by binding withTFPI160 are evenly distributed over the length of the peptide. Residuesundergoing the strongest perturbation of chemical shift were residuesS5, A9, Q11, Y28, and K29 with more than 4 ppm. Residues Y3, A4, V10,L12, S15, M21, A22, L23, and A24 were perturbed by more than 3 ppm.

Identification of Residues of JBT0303 that Interact with TFPI160

JBT0303 was produced recombinantly using the same procedure as describedabove for JBT0122 and isotope-labeled with ¹³C and ¹⁵N. The recombinantJBT0303 was named JBT0616 and had an additional glycine and serine atits N-terminus. The assignment of JBT0616 was performed on the basis ofHSQC, HNCACB and HNN spectra, which were recorded at 10° C. on a Varian500 MHz spectrometer and assigned using SPARKY software. The quality ofthe spectra of JBT0616 was better than that of JBT0788, although theexperimental conditions with respect to buffer, temperature, NMR tube,and NMR parameters were identical. The alpha carbon (CA), the betacarbon (CB), the amide proton (H), and the amide nitrogen (N) of mostresidues were assigned. The assignment was mainly based on the lesssensitive but more informative HNCACB instead of the HNCA. Incombination with the HNN spectrum, this resulted in an unambiguousassignment of all JBT0303 derived residues.

An assignment table for JBT0616 is provided in FIG. 51. The secondarystructure was extracted from Cα chemical shifts and determined by TALOSusing the assignments of H, CA, CB, CO and N. Like JBT0788, JBT0616exhibited a patch of positive Δδ(Cα) values indicative of α-helicalconformation. The helix was located at the C-terminal part of thepeptide and comprised residues 10-18. As for JBT0788, Δδ(Cα) values upto about 1.8 ppm were calculated, qualifying this helix as relativelystable for such a short peptide. A ribbon model illustrating thesecondary structure of JBT0616 is set forth in FIG. 52. The strongpositive value of the C-terminal residue 20 is, like residue 31 inJBT0788, typical for residues without a C-terminal neighbor. TheN-terminal residues 1-9 of JBT0616 exhibited slightly positive Δγ(Cc)values, suggesting a preference for an α-helical structure.

The assignment of JBT0616 in complex with TFPI160 was performed using a¹³C/¹⁵N-labelled peptide sample with an excess of unlabelled TFPI160.HSQC, HNCA, HNCOCA, and HNCO spectra were recorded on a Varian 800 MHzspectrometer and assigned using the SPARKY software. The spectra wererecorded at 30° C. Using these spectra, the alpha carbon (CA), the betacarbon (CO), the amide proton (H), and the amide nitrogen (N) of mostresidues were assigned, as set forth in the table in FIG. 53. Thesecondary structure of JBT0616 in complex with TFPI160 was extractedfrom Cαchemical shifts and calculated by TALOS Like the free peptide,JBT0616 in complex with TFPI160 exhibited a C-terminal patch of positiveΔδ(Cα) values indicative of α-helical conformation. The stability of theα-helix is increased upon complex formation. This finding suggests thatthe C-terminal region of JBT0616 is the core binding motif. The Δδ(Cα)values for the N-terminal residues also changed, but to a lesser extent.The secondary structure of JBT0616 when complexed with TFPI isillustrated in the ribbon model in FIG. 54.

The most significant changes of chemical shifts upon complex formationwere observed for residues Q2, K5, F8, V9 and A18 of JBT0616 with morethan 7 ppm. Residues F13, R17, K19 and L20 also were perturbed anddemonstrated chemical shift changes of more than 4 ppm. The strongchemical shift changes of residues at the N-terminus indicated that itis not only the amphipathic C-terminal α-helix which drives binding ofthe peptide to TFPI160.

Results from the NMR experiments in combination with analysis of JBT0477substitutions were used to create a model of KD1 in complex with JBT0303using HADDOCK (High Ambiguity Driven protein-protein DOCKing) software.HADDOCK is an information-driven flexible docking approach for themodeling of biomolecular complexes. HADDOCK distinguishes itself fromab-initio docking methods in the fact that it encodes information fromidentified or predicted protein interfaces in ambiguous interactionrestraints (AIRs) to drive the docking process. Identification of thebinding site on TFPI160 and the peptides as revealed by chemical shiftdata, the torsion angles of the peptides as determined by the softwareTALOS, and the substitution analysis of JBT0477 provide the restraintsfor the calculation of the models.

For the calculation of the KD1-JBT0303 HADDOCK models, the followingrestraints were employed: (a) torsion angles were taken from thecalculations of TALOS for K4, K5, V7, F8, Y12-A18 of JBT0303; (b)residues of KD1 with chemical shift changes of more than 1.5 ppm areinvolved in binding to JBT0303: F25, F28, D32, A37, I38, I46, F47, T48,F54 and Y56; (c) the hydrophobic side of the amphipathic helix ofJBT0303 is bound to KD1: Y12 or L16 or L20 of JBT0303 bind to D32 or A37or 138 or F54 or Y56 of KD1; (d) R15 or K19 of JBT0303 binds to D31 orD32 or E60 of KD1; (e) F8 or V9 of JBT0303 binds to F25 or F28 of KD1;(f) Y12 or F13 of JBT0303 binds to 146 or F47 or T48 of KD1; and (g) Q2of JBT0303 binds to Y56 of KD1. The Q2 JBT0303-Y56 KD1 interaction alsowas taken as a restraint for model calculation.

Strong chemical shift changes were observed for K5 of JBT0303 uponcomplex formation. For the remaining residues of JBT0303 considered todrive the peptide-protein interaction, the models are in good agreementwith the data. The model of KD1-JBT0303 with the lowest energy places F8of JBT0303 in proximity to F25 and F28 of TFPI, explaining the observedchemical shift changes and the data from the substitution analysis. V9of JBT0303 interacts with the hydrophobic patch of the KD1 includingF54. Y12, F13, L16 and L20 of JBT0303 also face the hydrophobic patch ofthe KD1. The proximity of Y12 to F28, I46, T48 of F13 to F47, T48, ofL16 to F54 and of L20 to A37, I38 causes the observed perturbations ofNMR chemical shift of those residues in the complex; the conservation ofY12 and L16 may be due to the extensive interactions of these residueswith the protein. K19 of JBT0303 is in a position allowing interactionwith D32 of KD1. The role of R15 of JBT0303 seems to be an interactionwith the hydrophobic patch of KD1 as well as with D32. Moreover, themodel explains why a negatively charged aspartate is preferred atposition 10 of JBT0303; it can interact with the positively charged K29of KD1. A glycine at position 11 of JBT0303 is present due to the stericand conformational restraints at this position. A HADDOCK model of KD1(TFPI residues 22-79 comprising KD1) in complex with JBT0303 is providedin FIG. 55.

Models of JBT0740 and JBT1857 Bound to KD1

Peptides JBT0740 and JBT1857 (FQSK-dP-NBHBDGYFERL-Aib-AKL (SEQ ID NO:178)), both derivatives of JBT0303, demonstrate significantly enhancedEC₅₀ values in the FXa-TFPI inhibition assay (0.11 μM and 0.0023 μM,respectively) and lower K_(d)'s as determined by Biacore. Models ofJBT0740 and JBT1857 in complex with TFPI KD1 (residues 22-79 of TFPI160)were calculated by HADDOCK using similar constraints as for JBT0303: (a)the constraints for the torsion angles of residues 4 and 5 of JBT0740and JBT1857 were amended in order to take account of the substitutionsat position 5 of the JBT0303 derivatives; (b) torsion angles were takenfrom the calculations of TALOS for V7, F8, Y12-A18 of JBT0303 and, incontrast to JBT0303, no fixed values for Phi and Psi were given for K4and for NmetG5/dP5; (c) NmetG5 and dP5 are in the cis conformation; (d)residues of KD 1 with chemical shift changes of more than 1.5 ppm areinvolved in binding to JBT0303: F25, F28, D32, A37, I38, I46, F47, T48,F54 and Y56; (e) the hydrophobic side of the amphipathic helix ofJBT0303 is bound to KD1; (f) Y12 or L16 or L20 of JBT0303 bind to D32 orA37 or 138 or F54 or Y56 of KD1; (g) residues R15 or K19 of JBT0303 bindto D31 or D32 or E60 of KD1; (h) residues F8 or V9 of JBT0303 bind toF25 or F28 of KD1; (i) residues Y12 or F13 of JBT0303 bind to 146 or F47or T48 of KD1; and (j) residue Q2 of JBT0303 binds to Y56 of KD1.

The energetically most favorable HADDOCK models of JBT0740 and JBT1857illustrated a different mode of binding compared to JBT0303. The mostobvious differences were in the region of residues 5 to 11. Lessdramatic deviations were observed at the N-terminus and the C-terminusof the peptides. However, the different binding of the termini alsomight contribute to the optimized binding of the JBT0303 derivatives toTFPI.

X-Ray Crystal Structure of JBT1857 Bound to KD1

The crystal structure of KD1 in complex with a KD1 binding peptide,JBT1857, was determined. TFPI was recombinantly expressed in E. coli andoxidatively refolded from inclusion bodies. TFPI amino acids 1-150comprising a thrombin cleavage site within the TFPI linker sequencejoining KD1 and KD2 (TFPI1-150-Thrombin(MADSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRDNANRLVPRGSQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG (SEQ ID NO: 4235)) was clonedinto an E. coli expression vector (pET19b). The TFPI 1-150-Thrombinsequence comprises two amino acids at the N-terminus that are artifactsof recombinant expression, and are not part of the wild-type TFPI aminoacid sequence. The sequences encoding Kunitz domain 1 and 2 are bolded.E. coli (BL21(DE3) pLysS) was cultivated in MagicMedia™ and TFPI1-150-Thrombin was expressed as insoluble inclusion bodies. Inclusionbodies were harvested by lysis of E. coli by incubation with BugBusterMaster Mix and purified upon washing with 50 mM Tris/HCl pH 8, 0.1%Tween 20. Inclusion bodies were dissolved in 8M urea, 50 mM Tris/HCl pH8.0 and TFPI 1-150-Thrombin was reduced upon addition of 20 mM DTT.Oxidative refolding was performed by rapid 1/10 dilution into a buffercontaining 50 mM Tris/HCl pH 10 and 1.1 mM oxidized Glutathion, followedby excessive dialysis against 20 mM Tris/HCl pH 7. RefoldedTFPI1-150-Thrombin was purified by a sequential purification protocolusing a Q Sepharose FF anion exchange and a peptide affinity (JBT131)media. Purified TFPI1-150-TFPI was proteolytically digested byincubation with thrombin (1 U thrombin/mg TFPI1-150-Thrombin, cleavagesite, LVPR/GS) resulting in the generation of Nterm KD1-Thrombin(MADSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIGGCEGNQNRFESLEECKKMCTRDNANRLVPR (SEQ ID NO: 4236)) and KD2-Thrombin(GSQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLE ECKNICEDG (SEQID NO: 4237)). Nterm KD1-Thrombin was purified from the digestionmixture using benzamidin sepharose for removal of thrombin, followed bya JBT131 peptide affinity column. Purified Nterm KD1-Thrombin was usedfor complex formation with JBT1857 and further crystallization.

The antagonistic peptide, JBT1857, was prepared by solid phasesynthesis. Successful co-crystallization of equimolar complexes wasobtained under 100 mM MES pH 6.5, 20% PEG 4000, 600 mM NaCl. Crystalsdiffracted to better than 2.5 Å resolution, albeit with somenon-merohedral twinning. Diffraction data were processed with iMosflmand SCALA from the CCP4 program package, revealing a monoclinic crystalform with unit cells dimensions of a=113.67 Å, b=69.32 Å, c=42.37 Å,α=90.0°, β=92.97°, γ=90.0°, spacegroup C2 (Leslie, Acta Crystallogr DBiol Crystallogr, 62(Pt 1), 48-57 (2006); Evans, Acta Crystallogr D BiolCrystallogr, 62(Pt 1), 72-82 (2006)). Self-rotation calculationsindicated an approximately two-fold non-crystallographic symmetry.Consistent herewith, two molecules were localized in the asymmetric unitrelated by a 170° rotation. The Patterson search was carried out byusing the program PHASER and a structure ensemble of the availableKunitz domain 2 crystal structures as search model (McCoy et al., J ApplCrystallogr, 40(Pt 4), 658-674 (2007)). The unit cell containedapproximately 64% solvent. Non-crystallographic electron densityaveraging and model building and model refinement was carried out withCoot, Refmac, MAIN and CNS programs. The current model was completelydefined for both copies of the JBT1857 peptide and the interaction withthe protein with current R=0.257, Rfree=0.298, deviation from idealgeometry rms(bond)=0.008 Å, rms(angle)=1.8°.

JBT1857 structure: The structure of JBT1857 can be segmented into (i)the N-terminal anchor consisting of acetylated Phe1_(AP)-Gln2_(AP); (ii)an Ω-shaped loop comprising Ser3_(AP)-Asn6_(AP); (iii) an intermediatesegment built from Val7_(AP) and His8_(AP); (iv) a tight glycine-loopcontaining Val9_(AP)-Gly11_(AP); and (v) the C-terminal α-helixcomprising Tyr12_(AP)-Leu20_(AP). As used herein, the subscript _(AP)indicates the sequence numbering in the “antagonistic peptide” JBT1857.The conformation of the α-helix is stabilized by a non-natural a-methylalanine positioned at the center of the helix (position 17_(AP)); aC-terminal amide that completes the 1-4 hydrogen bonding pattern of theα-helix; and a stacked cluster by the aromatic side chains of His8_(AP),Tyr12_(AP) and Phe13_(AP). These effects cooperate to stabilize theC-terminal α-helix spontaneously in solution, consistent with circulardichroism data on the peptide. The observed aromatic side chain stacking(His8_(AP), Tyr12_(AP), Phe13_(AP)) enforces a tight turn that can beonly accomplished by glycine at position 11_(AP). This structuralconstraint is reflected by dramatic losses in binding affinity uponreplacement of Gly11_(AP) by any other amino acid. The conformation ofthe N-terminal loop segment is partly stabilized by a D-proline, knownto induce a tight turn conformation, and a 1-4 hydrogen bond by thecarbonyl oxygen of Ser3 with the amide nitrogen of Asn6. All ring sidechains (Tyr1_(AP), Pro5_(AP), His8_(AP), Tyr12_(AP), Phe13_(AP)) pointtowards the same direction, enabling them to interact with the KD 1domain of TFPI.

Interaction of JBT1857 and KD1: The interactions between JBT1857 and KD1were determined. Hydrophobic contacts are interactions having anintermolecular distance of ≦4 Å, while hydrogen bonds have a distancebetween 2.6-3.2 Å. Phe1_(AP) interacts non-specifically with TFPI makingcontacts with Phe2 and Ala27. In contrast, Gln2_(AP) contacts a deeplyburied pocket of TFPI and makes hydrophobic interactions with Phe28,Lys29, Ile46 and Phe47. Moreover, the amide group of Gln2_(AP) formsthree H-bonds with Phe28-CO, Phe44-CO and Ile46-NH. The Ω-loop ofJBT1857, comprising Ser3-Asn6, mediates rather limited hydrophobicinteractions with the protein; Ser3, Pro5 and Asn6 interact with Lys29and Phe47. Val7_(AP) of JBT1857's intermediate segment also binds toLys29 and Phe47. His8 mainly contributes intramolecular aromaticstacking interactions with Tyr12_(AP) and partly with Phe13_(AP), andexhibits a hydrophobic interaction with Ala30 of TFPI. Similarly, theglycine-loop Val9_(AP)-Gly11_(AP) contributes few contacts with theKunitz domain. Val9_(AP) interacts directly with KD1 by forming ahydrogen bond with the carbonyl group of Ala30 and a hydrophobicinteraction with Asp32. Tyr12_(AP) mediates a hydrogen bond via itshydroxyl group with the amide nitrogen of Ile55 and a hydrophobicinteraction with Asp30. Leu16 is part of a hydrophobic contact withIle55. Beside the largely hydrophobic interactions of the C-terminalhelix of the peptide with the protein, there are electrostaticinteractions between Arg15_(AP) and Asp32. Furthermore, Lys19_(AP)contributes to binding with TFPI by forming a hydrogen bond to thecarbonyl group of Ala37 and contacts with Lys36 and Ile38. The TFPIcontact surface has an overall hydrophobic character with some chartedhot spots, and a driving force of complex formation with JBT1857 is thesteric surface complementarity.

This example describes characterization of the secondary structure ofexemplary peptides of the invention and correlates the structure withinhibitory function of the peptides. The example also identifies theTFPI amino acid residues that interact with JBT1857, a TFPI-bindingpeptide that inhibits TFPI activity.

Example 8

The following example describes additional TFPI-binding peptidesmodified by the addition of moieties that enhance physicochemical orpharmacokinetic properties of the peptides. The example furtherdescribes a method for assessing clot formation in whole blood usingrotation thromboelastography.

JBT1857 (JBT0047 peptide family) was conjugated to different PEGmoieties, and the binding affinity and TFPI inhibitory activity of thePEGylated peptides were examined. JBT1857 was modified by addition of aC-terminal cysteine to produce JBT2315 (Ac-FQSKpNVHVDGYFERL-Aib-AKLC-NH2(SEQ ID NO: 4077)), which was conjugated at the C-terminus with linearmaleimide PEG moieties of increasing size: 5 kD, 12 kD, 20 kD, 30 kD,and 40 kD, using the methods described in Example 5. The resultingPEGylated peptides were designated as follows:

TABLE 12 SEQ ID Peptide PEG (kD) Sequence NO JBT1857 —Ac-FQSKpNVHVDGYFERL-Aib-AKL-NH2 4020 JBT2317 —Ac-FQSKpNVHVDGYFERL-Aib-AKLC(NEM)-NH2 4078 JBT2325 5.3Ac-FQSKpNVHVDGYFERL-Aib-AKLC(PEG)-NH2 4086 JBT2326 12.1Ac-FQSKpNVHVDGYFERL-Aib-AKLC(PEG)-NH2 4087 JBT2327 21.0Ac-FQSKpNVHVDGYFERL-Aib-AKLC(PEG)-NH2 4088 JBT2328 29.1Ac-FQSKpNVHVDGYFERL-Aib-AKLC(PEG)-NH2 4089 JBT2329 41.5Ac-FQSKpNVHVDGYFERL-Aib-AKLC(PEG)-NH2 4090

Stability, Binding Affinity, and TFPI-Inhibitory Activity of PEGylatedPeptides

The PEGylated peptides demonstrated significantly increased plasmastability in mouse and human plasma. The peptides were added to samplesof mouse or human plasma, and the percentage of the initial amount ofpeptide remaining in plasma 24 hours after the addition was measured byIC₅₀ ELISA on Maxisorp plates coated with 0.05 mg/ml TFPI (2.26 nMtracer peptide JBT2271). Less than approximately 10% of the initialamount of JBT1857 and JBT2317 remained in plasma, while 40% or more ofthe initial amount of the PEGylated TFPI-binding peptides remained after24 hours. Approximately 60% or more of JBT2327 and JBT2329 was detected.PEGylated peptides also are significantly more stable in human plasmacompared to unmodified peptides. Approximately 60% or more of PEGylatedpeptide remained after 24 hours. The unmodified peptides were morestable in human plasma than mouse plasma; about 20% or more of theinitial amount remained after 24 hours of incubation.

The PEGylated peptides also were characterized in the assays describedin Examples 1-4 and compared to JBT1857. Representative results areprovided in Table 13 set forth below. The thrombin generation assay wasperformed as described in Example 4, and the results are provided asEC₅₀, corresponding to the concentration of peptide which improved peakthrombin (nM) half maximal.

TABLE 13 Thrombin Extrinsic generation in Competition FXa Tenase humanFVIII- Biacore ELISA Inhibition Inhibition inhibited plasma PEG K_(D)IC₅₀ EC₅₀ EC₅₀ EC₅₀ (kD) (nM) (nM) (nM) (nM) (nM) JBT1857 0.061 3.0 3.76.9 JBT2317 0.054 2.9 3.8 7.8 88 JBT2325 5.3 0.71 6.6 10.7 10.7 35JBT2326 12.1 1.1 9.3 9.3 9.3 34 JBT2327 21.0 1.3 10.9 7.2 7.2 24 JBT232829.1 1.6 12.3 6.0 6.0 19 JBT2329 41.5 1.1 12.6 6.0 12.8 19 *CompetitionELISA performed with tracer JBT2271 (1 nM) and 0.05 μg/ml TFPI in thecoating buffer.

Addition of the C-terminal cysteine blocked with NEM did notsignificantly influence the binding affinity of JBT2317 or the activityof the peptide in the FXa inhibition, extrinsic tenase assay, orthrombin generation assay compared to JBT1857. PEG size did notsignificantly impact the TFPI-binding peptides' ability to restoreactivity of FXa in the presence of TFPI-1. In the extrinsic tenase assayof Example 3, inhibitory activity increased with higher molecular weightPEG moieties up to 20 kD PEG. Activity did not further improve for 30 kDor 40 kD PEG moieties. In the thrombin generation assay of Example 4using human plasma, EC₅₀ decreased with PEG size, and maximal inhibitionof TFPI (as measured by peak FIIa (nM)) increased with PEG size. Inmouse plasma, attachment of 40 kD PEG to a TFPI binding peptideincreased maximal inhibition of TFPI.

The ability of PEGylated TFPI-binding peptides to restore extrinsictenase complex activity for converting FX to FXa also was determinedusing a cell-based extrinsic tenase assay using the method of Example 5.Addition of the C-terminal cysteine blocked with NEM did notsignificantly influence the activity of JBT2317 in the cell-basedextrinsic tenase assay compared to JBT1857. Conjugation of PEG moieties(5 kD, 20 kD, 30 kD, or 40 kD) to JBT2317 increased TFPI inhibitoryactivity by 5-20%.

Rotational Thromboelastography

Continuous visco-elastic assessment of human whole blood clot formationand firmness was performed by rotation thromboelastography with wholeblood preparations in the presence or absence of peptides. Blood samplesfrom a healthy individual were drawn into citrated Sarstedt Mono S(0.106 M or 3.2% (w/v) Na-citrate) (5 ml), mixing one part of citratewith nine parts blood, using a 21 gauge butterfly needle. A portion ofthe blood samples was incubated with high titer, heat-inactivatedanti-human FVIII antiserum raised in goat (3876 BU/ml; BaxterBioScience, Vienna, Austria) resulting in 51 BU/mL. Test samples wereprepared by dissolving quantities of peptides in either DMSO or HEPESbuffered saline (with or without 0.1% Tween 80).

Recordings were made using a ROTEM thromboelastography coagulationanalyzer (Pentapharm, Munich, Germany) at 37° C. Briefly, blood is addedinto a disposable cuvette in a heated cuvette holder. A disposable pin(sensor) is fixed on the tip of a rotating axis. The axis is guided by ahigh precision ball bearing system and rotates back and forth. The axisis connected with a spring for the measurement of elasticity. The exactposition of the axis is detected by the reflection of light on a smallmirror on the axis. The loss of elasticity when the sample clots leadsto a change in the rotation of the axis. The data obtained are computeranalyzed and visualized in a thromboelastogram. The thromboelastogramshows elasticity (mm) versus time (s). An elasticity of approximatelyzero is observed before clot formation begins. Mirror image traces aboveand below the zero line indicate the effect of clot formation on therotation of the axis.

Before starting each experiment, the citrated whole blood was mixed withcorn trypsin inhibitor (CTI) (Hematologic Technologies, Inc., EssexJunction, Vt., USA) providing a final concentration 62 μg/mL forspecific inhibition of FXIIa, in order to inhibit FXIIa-mediated contactactivation. The analytical set-up was as follows: to 20 μL of testsample or control, 300 μL of pre-warmed (37° C.) CTI treated citratedwhole blood was added, followed by 20 μL of a 1:15 dilution of TF PRPreagent containing recombinant human tissue factor (rTF, 3 pM) (TS40,Thrombinoscope BV, Maastricht, The Netherlands). Coagulation wasinitiated by the addition of 20 μL 200 mM CaCl₂ (star-TEM®, Pentapharm,Munich, Germany) and recordings were allowed to proceed for at least 120min. The final concentration of rTF in the assay was 11 or 44 fM.

The thromboelastographic parameters of clotting time (CT), clotformation time (CFT) and maximum clot firmness (MCF) were recorded inaccordance with the manufacturer's instructions. CT is defined as thetime from the start of measurement to the start of clot formation. CFTis defined as the time from the start of clot formation until anamplitude of 20 mm is reached. MCF is the maximum difference inamplitude between the two traces during the assay. The first derivativeof the data of the thromboelastogram are plotted to obtain a graph ofvelocity (mm/s) against time (s). From this graph, the maximum velocity(maxV) is determined. The time at which the maximum velocity is obtained(maxV-t) is also determined.

Exemplary results are illustrated in FIGS. 56 and 57. JBT1857 andJBT2317 restored coagulation parameters in Hem A blood. PEGylated (40kD) TFPI-binding peptide JBT2329 also restored prolonged coagulationparameters in Hem A blood, as illustrated in FIG. 57. PEGylation ofJBT2317 reduces clot time and clot formation time.

Nail Clip Study

The effect of JBT2329 on blood loss in naïve mice also was studied.C57BL6 mice were administered vehicle, 1 mg/kg JBT2329, or 0.1 mg/kgJBT2329 (N=19 or 20 for each group) intravenously 30 minutes prior tonail clip at 10 ml/kg. Animals were anaesthetized 10 minutes before nailclip with 80 mg/kg pentobarbital (i.p.). At time=0 minutes, the nail ofthe small toe of the right hind paw was cut just before the nail bed.The paw was transferred to a vial prefilled with 0.9% NaCl solution.Samples of blood were collected for analysis during the first 30 minutesfollowing the nail clip and the next 30 minutes thereafter, and meancollected volume for the groups was calculated and compared. Mean bloodloss in vehicle treated mice was about 30.5 μl over the first 30minutes, 52.1 over the second 30 minute period, resulting in about 82.6μl of blood loss over 60 minutes. In contrast, administration of 0.1mg/kg JBT2329 reduced blood loss by about 50% over the first 30 minutes(16.0 μl) and about 64% over the second 30 minute period (18.7 μl),resulting in about a 60% reduction of total blood loss over 60 minutes(34.7 μl) compared to vehicle-treated mice. Increasing the dose ofJBT2329 to 1.0 mg/kg further reduced blood loss by at least about 10%;12.2 μl was collected over the first 30 minutes, 10.6 μl was collectedover the second 30 minute period, resulting in 22.8 μl collected overthe entire 60 minute collection period. JBT2329 also efficiently reducedbleeding when administered subcutaneously compared to vehicle-treatednaïve mice; subcutaneous injection of 10 mg/kg JBT2329 reduced bloodloss during the 60 minutes following nail clip by approximately 58%compared to vehicle-treated subjects.

The results described above were generated using a JBT1857 derivativecomprising a linear PEG moiety attached to the C-terminus of the peptideand a JBT1586 derivative comprising a PEG moiety at the N-terminus.Peptides comprising an alternate conjugation site or alternativechemical moiety also were generated. A 40 kD linear PEG moiety wasconjugated to residue 14 of JBT1857 to generate JBT2404. The linear 40kD PEG moiety of JBT2329 was replaced with a 40 kD branched PEG moietyto generate JBT2401. JBT1857 also was modified to compriseK(Ttds-Maleimidopropionyl) (JBT2374) at the C-terminus. JBT2374 was usedto generate JBT2410, an HSA conjugate of JBT2374. JBT2375, aK(AOA)-comprising derivative of JBT1857, was used to couple PSA aldehydeto the peptide JBT1857, resulting in JBT2430. JBT2401, JBT2404, JBT2410and JBT2430 were characterized using the assays described above.Representative results are summarized in Table 14:

TABLE 14 Thrombin generation in human Human FVIII- ELISA FXa Plasmainhibited Biacore affinity Inhibition Stability, 24 plasma K_(D) EC₅₀EC₅₀ hour EC₅₀ (nM) (nM) (nM) (%) (nM) JBT2329 <1 12.6 6 67.3 1.4JBT2401 <1 22.4 7.7 85.1 1.4 JBT2404 <1 18.2 13.4 96.7 1.7 JBT2410 n.d.5.1 4.7 65.7 1.8 JBT2430 n.d. 5.6 9.0 135.6

This example demonstrates that an exemplary TFPI-binding peptide of theinvention, JBT1857, is a potent inhibitor of TFPI and can befunctionalized and conjugated with PEG without loss of activity.PEGylation increased TFPI-inhibitory activity in several functionalassays. Surprisingly, peptides conjugated to higher weight PEG moietiesdemonstrated enhanced TFPI inhibitory activity. JBT2329, comprising a 40kD linear PEG moiety, significantly reduced blood loss in aclinically-relevant animal model. PEG conjugation within the amino acidsequence of JBT1857, use of a branched PEG moiety, and attachment of HSAand PSA did not destroy the activity of the peptide.

Example 9

The following example describes the characterization of two TFPI-bindingpeptides of the invention, JBT1837 and JBT1857. JBT1837(Ac-SYYKWH[CAMRDMKGTMTC]VWVKF-NH) (SEQ ID NO: 1044) is a cyclic peptideof the JBT0120 family that binds KD1 and KD2 of TFPI. JBT1857(Ac-FQSKpNVHVDGYFERL-Aib-AKL-NH2) (SEQ ID NO: 178) is a linear peptideof the JBT0047 family that binds KD 1 of TFPI. The affinity andTFPI-inhibitory activity of JBT1837 and JBT1857 were examined using theassays described in Examples 1-4, the results of which are summarized inTable 15.

TABLE 15 Extrinsic Thrombin Thrombin FXa Tenase generation in generationin ELISA Inhibition; R&D Inhibition; R&D human FVIII- human FIX- Biacoreaffinity TFPI/flTFPI TFPI/flTFPI inhibited plasma deficient plasma K_(D)EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ (nM) (nM) (nM) (μM) (nM) (nM) JBT1837 <1 4.83.2/5.9 0.5/0.9 10 16 JBT1857 <1 3.0  3.7/21.9  6.9/13.6 69 51

Affinity of the peptides to human TFPI measured via BiaCore was lessthan 1 nM. Affinity measured by ELISA (IC₅₀) was 4.8 nM for JBT1837 and2.5 nM for JBT1857. JBT1837 dissociated from human TFPI more slowly thanJBT1857 (i.e., JBT1837 remained bound to human TFPI for a longer periodof time compared to JBT1857). A FXa inhibition assay was performed usingboth full length human TFPI (“flTFPI”) and truncated human TFPI (254amino acids “R&D TFPI”) (0.1 nM FXa, 0.5 nM TFPI, 0.25% DMSO). Activityof the truncated TFPI was fully inhibited by both JBT1837 and JBT1857 at0.5 nM TFPI, while full length TFPI was inhibited 85% and 95% by JBT1857and JBT1837, respectively. At higher concentrations of flTFPI (e.g., 10nM flTFPI), JBT1837 fully inhibited TFPI activity, while JBT1857partially inhibited TFPI activity. EC₅₀'s also were higher when flTFPIwas used in the FXa inhibition study.

In the extrinsic tenase assay, about 85% of truncated TFPI was inhibitedby both peptides. Full length TFPI activity was inhibited about 56% and48% by JBT1837 and JBT1857, respectively. Surprisingly, in thecell-based extrinsic tenase assay, JBT1837 inhibited the activitycell-associated TFPI by about 50% whereas JBT1857 almost fully inhibitedcell-bound TFPI activity. In the plasma-based functional assay, JBT1837inhibited TFPI more efficiently than JBT1857 in human FVIII-inhibitedplasma and FIX-deficient plasma. JBT1837 corrected blood coagulationparameters in FVIII-inhibited blood in the ROTEM assay described inExample 8. JBT1857 also positively impacted blood coagulationparameters, but performed less efficiently than JBT1837 in the assay.

This example compared the affinity and TFPI-inhibitory activity ofcyclic and linear TFPI-binding peptides that target different regions ofthe TFPI protein. JBT1837 (a cyclic peptide belonging to family JBT0120)and JBT1857 (a linear peptide belonging to family JBT0047) efficientlybind human TFPI with affinities less than 1 nM and are potentinhibitors. FXa-TFPI interaction is fully blocked at low TFPIconcentrations by both peptides, while TFPI inhibition by JBT1857 isreduced in the presence of higher concentrations of TFPI. Both peptidespartially inhibit the activity of full-length TFPI in the extrinsictenase assay, and JBT1857 inhibits TFPI activity to a greater degree inthe cell-based extrinsic tenase assay compared to JBT1837. Compared toJBT1857, JBT1837 more efficiently inhibits TFPI in FVIII-deficientplasma. Both peptides improve coagulation parameters of FVIII-inhibitedhuman whole blood by reducing clot time, while JBT1857 improves clotformation velocity to a lesser degree compared to JBT1837.

Example 10

This example illustrates the in vivo activity of TFPI-binding peptidesof the invention in a clinically-relevant animal model. As describedbelow, an exemplary TFPI-binding peptide significantly reduced bloodloss in an animal when administered with suboptimal doses of FVIII andFIX.

JBT2329, a PEGylated (40 kD) TFPI-binding peptide (JBT0047 family) thatcross-reacts with human and murine TFPI, was tested in tail-tip bleedingmodel in FVIII knock-out mice and FIX knock-out mice. FVIII knock-outmice closely minor the condition of hemophilia A patients, and thetail-tip bleeding model is widely used in research to assess efficacy ofdrugs by measuring, e.g., bleeding time, blood loss or survival. ADVATE,a commercially available rFVIII, served as a reference, and ADVATEbuffer-treated animals served as negative controls. Each group contained16 FVIII knock-out mice (8 female+8 male). JBT2329 (1 mg/kg or 0.1mg/kg) or anti-TFPI antibody (maTFPI; 18 mg/kg) was administered 30minutes before the tail-tip was cut. ADVATE (10 IU/kg or 50 IU mg/kg) orADVATE buffer was administered five minutes before the tail was cut off.Test and control substances were administered as an intravenous bolusvia a lateral tail vein injection. Animals were anaesthetized by anintraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine.Approximately 10 minutes later, 2 mm of the tail-tip was cut off. Thetail-tips were placed in warm saline (approximately 37° C.) and bloodwas collected over an observation period of 60 minutes. The amount ofblood was determined gravimetrically. At the end of the observationperiod of 60 minutes the animals were humanely killed by cervicaldislocation before recovery from anesthesia.

Median total blood loss in buffer-treated animals was 930 mg. Mediantotal blood loss in subjects treated with murine anti-TFPI antibody(maTFPI) was 724 mg. The reduction in median total blood loss was morepronounced when the subjects were administered maTFPI with ADVATE. Acombination of maTFPI+10 IU/kg ADVATE led to a median total blood lossof 136 mg, animals treated with maTFPI+50 IU/kg ADVATE experienced amedian total blood loss of 13 mg. Median blood losses of animals treatedwith either 10 or 50 IU/kg ADVATE alone experienced median blood loss of798 and 364 mg, respectively. The superiority of the combinationtreatment of maTFPI+ADVATE over ADVATE alone was statistically shown formaTFPI+50 IU/kg ADVATE versus 50 IU/kg ADVATE (p=0.0010). Although notstatistically significantly superior, blood loss in animals treated withmaTFPI+10 IU/kg ADVATE was distinctively lower than in animals treatedwith 10 IU/kg ADVATE alone.

Efficacy, defined as statistically significant superiority over bufferat a 2.5% level, was shown for JBT2329 dosed at 1 mg/kg in combinationwith 10 and 50 IU/kg ADVATE and dosed at 0.1 mg/kg in combination with50 IU/kg ADVATE (p<0.0004). Animals treated with JBT2329 in combinationwith ADVATE showed a clinically-relevant reduction in blood loss,although the results were not statistically significant (p≧0.0506).Administration of 1 mg/kg JBT2329 without ADVATE did not reduce mediantotal blood loss over that observed in buffer-treated animals (930 mg).

JBT2329 also was tested in a FIX knock-out tail-tip bleed mouse model,which is a clinically-relevant model for hemophilia B human patients.The methodology was substantially similar to that described above withrespect to the FVIII knock-out model. Instead of ADVATE, a recombinantFIX (rFIX) served as a reference. Median total blood loss inbuffer-treated animals was 935 mg. Median total blood loss in animalstreated with a murine anti-TFPI antibody (maTFPI) was 774 mg. Mediantotal blood loss was reduced further when the animals received combinedtreatment of maTFPI and rFIX. A combination of maTFPI+10 IU/kg rFIX ledto a median total blood loss of 25 mg, while animals treated withmaTFPI+50 IU/kg rFIX exhibited a median total blood loss of 10 mg.Median blood loss of animals treated with either 10 or 25 IU/kg rFIXalone experienced a median blood loss of 888 and 774 mg, respectively.

Efficacy, defined as statistically significant superiority over bufferat a 2.5% level, was shown for JBT2329 when dosed at 1 mg/kg incombination with 10 IU/kg rFIX and at 0.1 mg/kg in combination with 10IU/kg rFIX. The superiority of JBT2329 in combination with rFIX overadministration of rFIX alone was observed (p<0.0172), while treatmentwith 1 mg/kg JBT2329 alone did not lead to a significant reduction inmedian total blood loss compared with buffer-treated animals (p=0.321).

In summary, JBT2329 promoted a clinically-relevant reduction of bloodloss when co-administered with suboptimal doses of FVIII and rFIX at alldoses tested. Furthermore, intravenous administration of JBT2329 waswell tolerated in all subjects across all treatment groups without anysigns of acute toxicity.

Example 11

The TFPI-binding peptides described herein are suitable for detectingTFPI in a sample, such as biological sample. This example describes amethod for detecting TFPI using the inventive peptides in an ELISA-likeassay format.

The peptide sequence of JBT1857 was N-terminally modified by theaddition of a biotinyl-Ttds moiety to generate JBT2271(Biotinyl-Ttds-FQSKpNVHVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 4033)). A96-well microtiter plate (Maxisorp, Nunc) was coated with 50 μl per wellcoating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.3) containing a rangeof TFPI concentrations (0-3 μg/ml, human recombinant TFPI, R&D Systems)for 1 hour at room temperature. The plate was washed three times with350 μl/well wash buffer (175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1%Tween 80, pH 7.35). The plate was then blocked with 100 μl blockingbuffer (2% yeast extract, 175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1%Tween 80, pH 7.35) for 1 hour at room temperature. The plate was thenwashed three times with 350 μl wash buffer. Fifty μl of differentlyconcentrated JBT2271 solutions in wash buffer (100-0 nM) were added toeach well. The plate was incubated for 1 hour and washed three timeswith 350 μl wash buffer. To each well, 50 μl streptavidin-horseradishperoxidase conjugate (R&D Systems, 1:200 in wash buffer) is added. Afteran incubation period of 1 hour at room temperature, the plate was washedthree times with wash buffer. Fifty μl TMB solution (SeramunBlau fast,Seramun) was added to each well. After a 1.5 minute incubation at roomtemperature, the reaction was stopped by adding 50 μl 1 M H₂SO₄ perwell. Absorbance was measured in a photometer (Molecular DevicesSpectramax M5) at 450 and 620 nm.

JBT2271 allowed detection of as little as 4.1×10⁻¹⁴ mole of TFPI perwell. The results of the assay described above illustrate that theinventive peptides are powerful tools for identifying and/or quantifyingTFPI in a sample.

Example 12

This example describes conditions for an exemplary k_(off) assay forcharacterizing TFPI-binding peptides.

Wells of a microtiter plate (96 wells, Maxisorp, Nunc) are coated with1.6 nM TFPI in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.3) fortwo hours at room temperature. The plate is then washed three times with350 μl wash buffer (175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1% Tween 80,pH 7.35), and wells are blocked with 100 μl blocking buffer (2% yeastextract, 175 mM NaCl, 5 mM CaCl₂, 25 mM HEPES, 0.1% Tween 80, pH 7.35).If an incubation period of 24 hours is employed, the wells are blockedfor at least one hour. Control wells used for a 15 minute incubationperiod are blocked for an additional 23.5 hours.

For a 24 hour incubation period, the wells are washed three times with350 μl wash buffer and are incubated with 50 μl test peptide in washbuffer. The concentration of test peptide depends on the individual IC₉₀concentration determined in, e.g., the TFPI IC₅₀ ELISA assay describedherein. The TFPI-coated wells are exposed to test peptide forapproximately 15 minutes. The wells are subsequently washed three timeswith 350 μl wash buffer and 50 μl tracer peptide (competitor) is added.An exemplary tracer peptide is JBT2271 (1.13 nM in wash buffer). Controlwells (maximum signal) are incubated with tracer only. Blank wellslacking TFPI are incubated with tracer only. Addition of the tracerpeptide commences the 24 hour incubation period.

A 15 minute incubation period is employed as a control if the IC₉₀concentration of the test peptide leads to a 90% reduction of themaximum signal. Wells blocked for an additional 23.5 hours are washedthree times with 350 μl wash buffer to remove the blocking buffer.Subsequently, 50 μl analyte in wash buffer is added and the wells areincubated for 15 min. The concentration of test peptide utilized dependson the peptide's IC₉₀ concentration determined using, e.g., a TFPI IC₅₀ELISA assay. The 15 minute incubation is followed by three washes with350 μl wash buffer and addition of 50 μl tracer peptide. Control wells(maximum signal) are incubated with tracer only. Blank wells lackingTFPI also are incubated with tracer only.

The plate is washed three times with 350 μl wash buffer, and 50 μlstreptavidin-horseradish peroxidase conjugate (R&D Systems, 1:200 inwash buffer) is added to each well. After an incubation period of onehour at room temperature, the plate is washed three times with washbuffer. TMB solution (50 μl per well; SeramunBlau fast, Seramun) isadded. After a 1.5 minute incubation at room temperature, the reactionis stopped by the addition of 50 μl 1 M H₂SO₄ per well. Absorbance ismeasured using a photometer (Spectramax M5, Molecular Devices) at 450and 620 nm. The assay results are presented as a percentage of thecorrected optical density (OD450-OD620) of wells exposed to test peptideand tracer peptide in relation to TFPI-coated wells exposed only totracer.

Example 13

TFPI inhibits FVIIa/TF activity by binding to FVIIa via Kunitz domain 1(KD1). This example describes an exemplary method for evaluating theinfluence of TFPI-binding peptides on TFPI's inhibition of FVIIa/TF.

Kinetic measurements were performed in 25 mM HEPES, 175 mM NaCl, 5 mMCaCl₂, 0.1% BSA, pH 7.3 at 25° C. in 96-well microtiter plates. Twentyμl soluble tissue factor (residues 33-251; Creative Biomart) and 20 μlFVIIa (ERL) at final concentrations of 100 nM and 5 nM, respectively,were mixed and incubated for 15 minutes. Twenty μl of TFPI-bindingpeptide in varying final concentrations (0-2 μM) were added to themixture and incubated for a further 15 minutes. In order to measure theresidual activity of the FVIIa/sTF complex, the reaction mixture wasincubated for 60 minutes with 20 μl TFPI (200 nM). The reaction wasinitiated by the addition of a chromogenic substrate, Chromozym-tPA(Roche) (1 mM). The change in absorbance at 405 nm was monitored byusing a Labsystems iEMS ELISA Reader for 30 minutes. FVIIa/sTF activitymeasured in the absence of TFPI was considered “100% activity” in thecontext of the assay. By plotting peptide concentration against residualactivity, EC₅₀ values were determined.

JBT1857 and JBT1837 were screened against TFPI160, TFPI1-150-Thrombin,NTermKD1, KD1, and KD2 (negative control). JBT1857 demonstrated an EC₅₀of approximately 0.21-0.23 μM for TFPI160, TFPI1-150-Thrombin, NTermKD1,and KD1. JBT1837, which binds KD1 and KD2, demonstrated an EC₅₀ ofapproximately 0.17-0.19 μM for TFPI160 and TFPI1-150-Thrombin, whileactivity in assays involving NTermKD1 and KD 1 was approximatelybackground.

The results described above demonstrate that TFPI-binding peptidesefficiently inhibit TFPI-FVIIa/TF interaction. JBT1857 efficientlyinhibited TFPI fragments containing KD1 as a minimal functional entity.Thus, this enzymatic assay confirms X-ray crystallographic data placingthe binding site of JBT1857 within KD1. JBT1837 inhibited TFPI fragmentscontaining the first two Kunitz domains, suggesting that the JBT1837binding site(s) are located within KD1-linker-KD2 region of TFPI. Acombination of Kunitz domains and fragments of a thrombin cleaved TFPI(1-150) did not restore inhibitory activity of JBT1837 in thechromogenic assay. The enzymatic assay described herein is a suitablesurrogate for detecting binding of a TFPI-binding peptide (or a testcompound) to TFPI, and is useful for examining the TFPI-inhibitoryeffect of TFPI-binding compounds.

Example 14

This example describes the influence of PEG and HSA conjugation onexemplary TFPI-binding peptides in vivo.

For pharmacokinetic analysis, C57B16 mice were treated with variousTFPI-binding peptides conjugated to different molecular weight PEGs andHSA. The dose of the peptide-PEG and peptide-HSA conjugates wasnormalized to 1 mg/kg (peptide content). Normalization assurescomparability between the conjugates of different molecular weight. Thepeptide conjugates were dissolved in 175 mM NaCl, 25 mM HEPES pH 7.35and administered intravenously via the tail vein or subcutaneously inthe neck region. Blood draws were taken from three animals (retrobulbar) and collected in heparinized vials at several time pointsfollowing administration. The samples were centrifuged, and thepeptide-conjugate content in plasma was quantified by ELISA.

FIG. 63 illustrates the concentration of PEGylated TFPI-peptidesdetected in plasma at several time points following administration, andTable 16 provides detailed information about the terminal half life andbioavailability of JBT2325-JBT2329, JBT2401, JBT2404 and JBT2410.

TABLE 16 JBT2325 JBT2326 JBT2327 JBT2328 JBT2329 JBT2401 JBT2404 JBT2410HL_λ_z [h] 0.16 0.35 4.2 10.1 19.8 20.7 12.3 7.8 (intravenous)Bioavailability [%] 58.2 76.0 89.7 52.0 73.3 58.4 59.3 46.6 (s.c.)

JBT2329, JBT2401 and JBT2404 are peptides conjugated to 40 kDa linearPEG (JBT2329 and JBT2404) or 40 kDa branched PEG (JBT2401). The 40 kDaconjugates exhibited a longer terminal half-life (HL_λ_z) compared topeptides conjugated to smaller PEGs following intravenousadministration. The area under curve (AUC) of the concentration-timecurve resulting from subcutaneous administration of the peptides wascompared to the AUC generated following intravenous administration tocalculate the bioavailability of the peptides. Results are shown inTable 16. The data demonstrate that TFPI-binding peptide conjugation tohigher molecular weight molecules allows a subcutaneous bioavailabilityof more than 30%.

FIGS. 64A-C illustrate the pharmacokinetic profile of JBT2401, JBT2404,and JBT2410 resulting from subcutaneous and intravenous administrationof the peptides to mice. JBT2404 comprises a PEG conjugated to cysteinein position X4014 relative to formula (XI). JBT2401 comprises a branchedPEG, and JBT2410 is conjugated to HSA. FIG. 64A demonstrates that fusionof a higher molecular weight molecule to a TFPI-binding peptide at aninternal position increases half life. Half life also is increased ifusing branched PEG (JBT2401) and HSA, which increased the in vivo halflife of JBT2410 compared to conjugates having smaller-sized PEGs (e.g.,JBT2325) or free peptide (see FIG. 31).

This example illustrates that the in vivo properties of various peptidesdescribed herein can be improved by conjugation with higher molecularweight molecules (like PEG) and/or with nFcR ligands (like HSA).

1.-60. (canceled)
 61. A method for identifying a TFPI-binding compound,the method comprising (a) contacting a peptide comprising TFPI Kunitzdomain 1 (KD1) with a test compound, and (b) detecting binding of thetest compound to a TFPI binding site defined by KD 1 amino acid residuescorresponding to human TFPI residues Phe28, Lys29, Ala30, Asp32, Ile46,Phe47, and Ile55.
 62. The method of claim 61, wherein the binding siteis defined by amino acid residues corresponding to human TFPI residuesAla27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47,and Ile55.
 63. The method of claim 62, wherein the binding site isdefined by amino acid residues corresponding to human TFPI residuesAla27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44,Ile46, Phe47, and Ile55. 64.-72. (canceled)
 73. A method of identifyinga TFPI inhibitor, the method comprising (a) contacting a peptideconsisting essentially of TFPI Kunitz domain 1 (KD1) with FXa in thepresence of a test compound under conditions that allow binding of KD1to FXa, and (b) comparing KD1-FXa binding in step (a) with KD1-FXabinding in the absence of the test compound, wherein a decrease inKD1-FXa binding in the presence of the test compound compared to KD1-FXabinding in the absence of the test compound indicates that the testcompound is a TFPI inhibitor.
 74. A composition comprising a TFPIinhibitor identified by the method of claim
 58. 75.-81. (canceled)
 82. Amethod for inhibiting human TFPI, the method comprising contacting humanTFPI with an inhibitor that binds human TFPI at a binding site definedby amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, andIle55.
 83. A method for treating a subject suffering from a disease orat risk of suffering from a disease, the method comprising administeringto the subject an inhibitor that binds human TFPI at a binding sitedefined by amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47,and Ile55.
 84. The method of claim 82, wherein the human TFPI bindingsite is defined by amino acid residues Ala27, Phe28, Lys29, Ala30,Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55.
 85. The method ofclaim 84, wherein the human TFPI binding site is defined by amino acidresidues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38,Phe44, Ile46, Phe47, and Ile55. 86.-87. (canceled)
 88. A computerstorage media having computer executable instructions that, whenexecuted on the processor of a computer, implement a method of modelinginteraction between selected three dimensional (3D) points in a TFPIKunitz domain 1 (KD1) protein and a test compound, the methodcomprising: obtaining a protein structure 3D model for the TFPI KD1protein; determining a 3D relationship between a selected subset ofamino acids in the protein structure, wherein the selected subset ofamino acids comprises Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, andIle55; modeling a surface bounded by the selected subset of amino acids;obtaining a test compound 3D model of a test compound; matching the testcompound 3D model to the surface bounded by the selected subset of aminoacids; and identifying contact points between the selected subset ofamino acids of the surface and the test compound 3D model. 89.-99.(canceled)
 100. A method of comparing a test compound to selected threedimensional points in a TFPI Kunitz domain 1 (KD1) protein, the methodcomprising: creating a protein structure for the KD1 protein in a memoryof a computer; determining a three dimensional model of a selectedsubset of amino acids in the KD1 protein at a processor of the computer,wherein the selected subset of amino acids comprises Phe28, Lys29,Ala30, Asp32, Ile46, Phe47, and Ile55; determining a three dimensionalmodel of a test compound at the processor of the computer; fitting the3D model of the test compound to the 3D model of the selected subset ofamino acids at the processor of the computer; and generating an affinityof the test compound for the selected subset of amino acids at theprocessor of the computer, wherein the affinity is based on a number ofamino acids in the subset in contact with the test compound and a bondstrength at each contact point. 101.-104. (canceled)
 105. A computerstorage media having computer executable instructions that, whenexecuted on the processor of a computer, implement a method of comparinga peptide to selected three dimensional points (3D) in a TFPI Kunitzdomain 1 protein (KD1), the method comprising: creating a proteinstructure for the KD1 protein; determining a three dimensional model ofa selected subset of amino acids in the KD1 protein, wherein the subsetof amino acids comprises Phe28, Lys29, Ala30, Asp32, Ile46, Phe47 andIle55; determining a three dimensional model of a peptide; fitting the3D model of the peptide to the 3D model of the selected subset of aminoacids; and generating an affinity of the peptide for the selected subsetof amino acids, wherein the affinity is based on a number of amino acidsin the subset in contact with the peptide and a bond strength at eachcontact point. 106.-107. (canceled)
 108. The method according to claim83, wherein the human TFPI binding site is defined by amino acidresidues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46,Phe47, and Ile55.
 109. The method according to claim 108, wherein thehuman TFPI binding site is defined by amino acid residues Ala27, Phe28,Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47,and Ile55.
 110. The method according to claim 83, wherein the disease isa blood coagulation disorder.
 111. The method according to claim 83,wherein the inhibitor is a peptide.
 112. The method according to claim111, wherein the peptide is conjugated to a polyethylene glycol (PEG)moiety.
 113. The method according to claim 111, wherein the peptide isconjugated to human serum albumin (HSA), an antibody or fragmentthereof, hydroxyethyl starch, a proline-alanine-serine multimer(PASylation), a C12-C18 fatty acid, or polysialic acid.